Targeting the glutamine to pyruvate pathway for treatment of oncogenic kras-associated cancer

ABSTRACT

Methods and kits for GPP-targeting, e.g., for the treatment of oncogenic Kras-associated cancers, and methods for determining the efficacy of those methods are provided.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research described in this application was supported in part bygrants (Nos. T32 CA009382-26, P01 CA117969, and 5P01CA120964-05) fromthe National Institutes of Health and grant no. R01 CA157490 from theNational Cancer Institute. Thus, the government has certain rights inthe invention.

TECHNICAL FIELD

Methods and kits for -targeting the glutamine to pyruvate pathway (GPP),e.g., for the treatment of oncogenic Kras-associated cancers, andmethods for determining the efficacy of those methods are provided.

BACKGROUND

Oncogenic mutant Kras signaling drives uncontrolled proliferation andenhances survival of cancer cells through the activation of itsdownstream signaling pathways, such as the MAPK and PI3K-mTOR pathways.To meet the increased anabolic needs of enhanced proliferation, cancercells require both sufficient energy and biosynthetic precursors ascellular building blocks to fuel cell growth. Under normal conditions,differentiated cells primarily metabolize glucose through themitochondrial tricarboxylic acid (TCA) cycle to drive the production ofATP to sustain basic cellular functions. In cancer cells, metabolicpathways are rewired in order to divert nutrients, such as glucose andglutamine, into anabolic pathways to satisfy the demand for cellularbuilding blocks. Accumulating evidence indicates that the reprogrammingof tumor metabolism is under the control of various oncogenes andoncogenic signals. The Ras oncogene in particular has been shown topromote glycolysis. However, the mechanisms by which oncogenic Krascoordinates the shift in metabolism to sustain tumor growth,particularly in the tumor microenvironment, and whether specificmetabolic pathways are essential for Kras-mediated tumor maintenanceremain areas of active investigation.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancerswith a 5 year survival rate of 3%-5%. Malignant progression frompancreatic intraepithelial neoplasia (PanINs) to highly invasive andmetastatic disease is accompanied by the early acquisition of activatingmutations in the KRAS oncogene, which occurs in greater than 90% ofcases, and subsequent loss of tumor suppressors including Ink4a/Arf, p53and Smad4. The high mortality rate of PDAC can be attributed to severalfeatures; namely the advanced stage of presentation and its profoundresistance to all forms of therapy, including conventional chemotherapy,targeted agents, and radiotherapy. Thus, there is a strong impetus toidentify new therapeutic targets for this disease.

SUMMARY OF THE INVENTION

As follows from the Background section above, there remains a need inthe art for methods for treating or preventing oncogenic Kras-associatedcancers. Such methods, as well as other, related benefits, are presentlyprovided, as discussed in detail below.

Cancer cells are characterized by metabolic dependencies thatdistinguish them from their normal counterparts and, in some cancers,glutamine (Gln) is used to fuel anabolic processes. The presentdisclosure describes the identification of a non-canonical pathway ofGln utilization in human pancreatic ductal adenocarcinoma (PDAC) cellsthat is required for tumor growth. While most cells utilize glutamatedehydrogenase (GLUD1), which converts Gln-derived glutamate intoα-ketoglutarate in the mitochondria to fuel the tricarboxylic acid (TCA)cycle, it is presently discovered that PDAC rely on a distinct pathway(termed, herein, the “glutamine to pyruvate pathway” or “GPP”) that isinitiated with the conversion of Gln to aspartate followed by theconversion of Gln-derived aspartate into oxaloacetate (OAA) in thecytoplasm. Subsequently, this OAA is converted into malate, whichenables the shuttling of NADH into the mitochondria for oxidativephosphorylation. The malate is then converted into pyruvate yieldingcytosolic NADPH to maintain the cellular redox state. Importantly, PDACcells are strongly dependent on this series of reactions, as Glndeprivation or genetic inhibition of any enzyme in this pathway led toan increase in reactive oxygen species and a reduction of reducedglutathione. Moreover, unlike normal pancreatic ductal cells, knockdownof any component enzyme in this series of reactions also results in apronounced suppression of PDAC growth in vitro and in vivo. Furthermore,it is demonstrated herein that the reprogramming of Gln metabolism ismediated by oncogenic Kras, the signature genetic alteration in PDAC,via the transcriptional upregulation and repression of key metabolicenzymes in this pathway. The discovery of the essentiality of thispathway in PDAC and the fact that it is dispensable in normal cells thusprovide novel therapeutic approaches to treat these refractory tumors aswell as other oncogenic Kras-associated cancers.

Thus, in certain aspects, a method for determining the efficacy ofGPP-targeting in a subject comprising an oncogenic Kras mutation (e.g.,a subject with a cancer associated with an oncogenic Kras mutation), andthe GPP-targeting comprises targeting an enzyme (e.g., Kras, GLS, GOT1,GOT2 and MDH1) or metabolite associated with an enzyme-catalyzedreaction in the GPP that is upstream of the malic enzyme (ME1)-catalyzedreaction (e.g., with an inhibitor, e.g., an anti-sense oligonucleotides,shRNA, siRNA, intrabodies, or small molecule, of an enzyme or metaboliteassociated with an enzyme-catalyzed reaction in the GPP), the methodcomprising: determining the level of one or more markers selected fromthe group consisting of NADP+, NADPH, GSSG, GSH, pyruvate, and reactiveoxygen species (ROS) (e.g., hydrogen peroxide, super oxide, hydroxylradical, hypochlorous acid, nitric oxide, peroxyl radical, and singletoxygen), in a sample obtained from a subject who is undergoing or hasundergone the GPP-targeting; and concluding that the GPP-targeting waseffective if the level of one or more of the markers NADP+, GSSG and ROSis increased, relative to each marker's control level, or if the levelof one or more of the markers NADPH, GSH and pyruvate is decreased,relative to each marker's control level; or concluding that theGPP-targeting was not effective if the level of one or more of themarkers NADP+, GSSG, and ROS is not increased, relative to each marker'scontrol level, or if the level of one or more of the markers NADPH, GSHand pyruvate is not decreased, relative to each marker's control level.In some aspects, the method comprises determining the level of two ormore, three or more, four or more, five or more, or all of the markersselected from the group consisting of NADP+, NADPH, GSSG, GSH, pyruvate,and ROS.

Preferably, each marker's control level is the level of the marker in asample obtained from the same subject prior to or at the beginning ofthe GPP-targeting or from another subject who is known to have a cancerassociated with an oncogenic Kras mutation and is not undergoing or hasnot undergone GPP-targeting. The control level of the marker may also bea predetermined reference level of the marker.

In other aspects, the method for determining the efficacy ofGPP-targeting further comprises determining the level of at least oneadditional marker selected from the group consisting of glutamine,glutamate, aspartate, αKG, NAD+, NADH, oxaloacetate, malate, MDH, andME1.

In one aspect, the GPP-targeting comprises inhibiting the enzyme GOT2,and the method comprises concluding that the GPP-targeting was effectiveif the level of one or more of the markers NADP+, GSSG and ROS isincreased, relative to each marker's control level, or if the level ofone or more of the markers NADPH, GSH, and pyruvate is decreased,relative to each marker's control level; and if the level of at leastone of the markers selected from the group consisting NAD+ and NADH isaltered (i.e., increased or decreased) relative to each marker's controllevel; or concluding that the GPP-targeting was not effective if thelevel of one or more of the markers NADP+, GSSG and ROS is notincreased, relative to each marker's control level, or if the level ofone or more of the markers NADPH, GSH, and pyruvate is not decreased,relative to each marker's control level; and if the level of at leastone of the markers selected from the group consisting of NAD+ and NADHis not altered relative to each marker's control level.

In another aspect, the GPP-targeting comprises inhibiting the enzymeKras, and the method comprises concluding that the GPP-targeting waseffective if the level of one or more of the markers NADP+, GSSG and ROSis increased, relative to each marker's control level, or if the levelof one or more of the markers NADPH, GSH, and pyruvate is decreased,relative to each marker's control level; and if the level of at leastone of the markers selected from the group consisting of glutamine,oxaloacetate, and NAD+ is increased relative to its control level; or ifthe level of at least one of the markers selected from the groupconsisting of aspartate, αKG, malate, MDH1, and ME1 is decreasedrelative to each marker's control level; or concluding that theGPP-targeting was not effective if the level of one or more of themarkers NADP+, GSSG and ROS is not increased, relative to each marker'scontrol level, or if the level of one or more of the markers NADPH, GSH,and pyruvate is not decreased, relative to each marker's control level;and if the level of of at least one of the markers selected from thegroup consisting of glutamine, oxaloacetate, and NAD+ is not increasedrelative to its control level; or if the level of at least one of themarkers selected from the group consisting of aspartate, αKG malate,MDH1, and ME1 is not decreased relative to each marker's control level.

In yet another aspect, a method for determining the efficacy ofoncogenic Kras inhibition in a subject comprising a cell having anoncogenic Kras mutation is provided, the method comprising: determiningthe expression level of at least one of the enzymes MDH1 and ME1 in asample obtained from a subject who is undergoing or has undergone theoncogenic Kras inhibition; and concluding that the oncogenic Krasinhibition was effective if the expression level of the at least oneenzyme is decreased compared to a control level of the enzyme; orconcluding that the oncogenic Kras inhibition was not effective if theexpression level of the at least one enzyme is not decreased compared toa control level of the enzyme.

In another aspect, the GPP-targeting comprises inhibiting the enzymeGOT1, and the method concluding that the GPP-targeting was effective ifthe level of one or more of the markers NADP+, GSSG and ROS isincreased, relative to each marker's control level, or if the level ofone or more of the markers NADPH, GSH, and pyruvate is decreased,relative to each marker's control level; and if the level of at leastone of the markers selected from the group consisting of aspartate, αKG,and NAD+ is increased relative to each marker's control level; or if thelevel of at least one of the markers selected from the group consistingof oxaloacetate, malate, glutamate, and NADH is decreased relative toeach marker's control level; or concluding that the GPP-targeting wasnot effective if the level of one or more of the markers NADP+, GSSG andROS is not increased, relative to each marker's control level, or if thelevel of one or more of the markers NADPH, GSH, and pyruvate is notdecreased, relative to each marker's control level; and if the level ofat least one of the markers selected from the group consisting ofaspartate, αKG, and NAD+ is not increased relative to each marker'scontrol level; or if the level of at least one of the markers selectedfrom the group consisting of oxaloacetate, malate, glutamate, and NADHis not decreased relative to each marker's control level.

In another aspect, the GPP-targeting comprises inhibiting the enzymeMDH1, and the method includes concluding that the GPP-targeting waseffective if the level of one or more of the markers NADP+, GSSG and ROSis increased, relative to each marker's control level, or if the levelof one or more of the markers NADPH, GSH, and pyruvate is decreased,relative to each marker's control level; and if the level of at leastone of the markers selected from the group consisting of oxaloacetateand NAD+ is increased relative to each marker's control level; or if thelevel of at least one of the markers selected from the group consistingof malate and NADH is decreased relative to each marker's control level;or concluding that the GPP-targeting was not effective if the level ofone or more of the markers NADP+, GSSG and ROS is not increased,relative to each marker's control level, or if the level of one or moreof the markers NADPH, GSH, and pyruvate is not decreased, relative toeach marker's control level; and if the level of at least one of themarkers selected from the group consisting of oxaloacetate and NAD+ isnot increased relative to each marker's control level; or if the levelof at least one of the markers selected from the group consisting ofmalate and NADH is not decreased relative to each marker's controllevel.

In another aspect, the GPP-targeting comprises inhibiting the enzymeGLS, and the method comprises concluding that the GPP-targeting waseffective if the level of one or more of the markers NADP+, GSSG and ROSis increased, relative to each marker's control level, or if the levelof one or more of the markers NADPH, GSH, and pyruvate is decreased,relative to each marker's control level; and if the level of at leastone of the markers selected from the group consisting of glutamine andNAD+ is increased relative to each marker's control level, or if thelevel of at least one of the markers selected from the group consistingof aspartate, oxaloacetate, malate, glutamate, and NADH is decreasedrelative to each marker's control level; or concluding that theGPP-targeting was not effective if the level of one or more of themarkers NADP+, GSSG and ROS is not increased, relative to each marker'scontrol level, or if the level of one or more of the markers NADPH, GSH,and pyruvate is not decreased, relative to each marker's control level;and if the level of at least one of the markers selected from the groupconsisting of glutamine and NAD+ is not increased relative to eachmarker's control level, or if the level of at least one of the markersselected from the group consisting of aspartate, oxaloacetate, malate,glutamate, and NADH is not decreased relative to each marker's controllevel.

In yet other aspects, a method for determining the efficacy ofGPP-targeting in a subject comprising an oncogenic Kras mutation, andthe GPP-targeting comprises targeting one or more of the enzymescatalyzing the conversion of glutamine to aspartate, the methodcomprising: determining the level of at least one marker selected fromthe group consisting of GSSG, GSH, aspartate, αKG, NADP+, NADPH,mitochondrial NAD+, mitochondrial NADH, pyruvate, oxaloacetate, andmalate in a sample obtained from a subject who is undergoing or hasundergone the GPP-targeting; and concluding that the GPP-targeting waseffective if the level of one or more of the markers GSSG, GSH,aspartate, αKG, NADP+, NADPH, NAD+, NADH, pyruvate, oxaloacetate, andmalate is altered relative to each marker's control level. Preferablythe one or more of the enzymes catalyzing the conversion of glutamine toaspartate is selected from GLS and GOT2.

In another aspect, the method includes concluding that the GPP-targeting(e.g., with an inhibitor of GOT1 or MDH1) was not effective if the levelof aspartate is not increased relative to a control level; or concludingthat the GPP-targeting was effective if the level of aspartate isincreased relative to a control level.

Also provided herein is a method for determining the efficacy ofglutamine to pyruvate pathway (GPP)-targeting in a subject having anoncogenic Kras mutation, and the GPP-targeting comprises inhibitingGOT1, MDH1, or ME1, the method including: determining the level ofaspartate in a sample obtained from a subject who is undergoing or hasundergone the GPP-targeting; and concluding that the GPP-targeting waseffective if the level of aspartate is increased relative to a controllevel; or concluding that the GPP-targeting was not effective if thelevel of aspartate is not increased relative to a control level.

In still other aspects, a method for treating cancer in a subjectcomprising a cancer cell expressing an oncogenic Kras mutation isprovided, the method comprising administering to the subject atherapeutically effective amount of a composition comprising aninhibitor of the enzyme ME1. In certain aspects, the method furthercomprises administering a therapeutically effective amount of acomposition comprising an inhibitor that targets an additional enzymeassociated with an enzyme-catalyzed reaction in the GPP or that targetsa metabolite associated with an enzyme-catalyzed reaction in the GPP.The additional enzyme may be selected from the group consisting of,e.g., Kras, GLS, GOT2, GOT1 and MDH1. In preferred aspects, theinhibitor of the metabolite targets a metabolite selected from the groupconsisting of glutamine, glutamate, aspartate, GSH, NADH, NADPH,oxaloacetate, and malate.

In another aspect, a method for treating cancer in a subject comprisinga cancer cell expressing an oncogenic Kras mutation is provided, themethod comprising administering to the subject: a therapeuticallyeffective amount of an inhibitor of the enzyme GLS or GOT2; and atherapeutically effective amount of a composition comprising aninhibitor that targets an additional enzyme associated with anenzyme-catalyzed reaction of the GPP or that targets a metaboliteassociated with an enzyme-catalyzed reaction in the GPP. Preferably, theinhibitor of the additional enzyme or metabolite targets one or more ofthe enzymes selected from the group consisting of Kras, GOT1, MDH1, andME1, or a metabolite selected from the group consisting of glutamine,glutamate, aspartate, GSH, NADH, NADPH, oxaloacetate, and malate.

In still other aspects, methods for preventing cancer in a subjectcomprising an oncogenic Kras mutation are provided. Preferably, themethods comprise administering to the subject a therapeuticallyeffective amount of a composition comprising an inhibitor of the enzymeME1. In certain aspects, the method further comprises administering atherapeutically effective amount of a composition comprising aninhibitor that targets an additional enzyme associated with anenzyme-catalyzed reaction of the GPP or that targets a metaboliteassociated with an enzyme-catalyzed reaction in the GPP. In preferredaspects, the additional enzyme is selected from the group consisting ofKras, GLS, GOT1 and MDH1.

In another aspect, a method for preventing cancer in a subjectcomprising an oncogenic Kras mutation is provided, wherein the methodcomprises administering to the subject a therapeutically effectiveamount of an inhibitor of the enzyme GLS or GOT2; and a therapeuticallyeffective amount of a composition comprising an inhibitor that targetsan additional enzyme associated with an enzyme-catalyzed reaction of theGPP or that targets a metabolite associated with an enzyme-catalyzedreaction in the GPP. Preferably, the inhibitor of the additional enzymeor metabolite targets one or more of the enzymes selected from the groupconsisting of Kras, GOT1, MDH1, and ME1, or a metabolite selected fromthe group consisting of glutamine, glutamate, aspartate, GSH, NADH,NADPH, oxaloacetate, and malate.

In certain aspects kits are provided for use in GPP-targeting, e.g., forthe treatment of a cancer associated with an oncogenic Kras mutation.For example, a kit comprising an inhibitor of ME1 and one or moreinhibitors of one or more of the enzymes selected from the groupconsisting of Kras, GOT2, GLS, GOT1, and MDH1 is provided. In anotheraspect, a kit comprising an inhibitor of GLS and one or more inhibitorsof one or more of the enzymes selected from the group consisting ofKras, GOT2, ME1, GOT1, and MDH1 is provided. In yet another aspect, akit comprising an inhibitor of at least one of the enzymes selected fromthe group consisting of Kras, ME1, GLS, GOT1, GOT2, and MDH1, and aninhibitor of one or more metabolites associated with an enzyme-catalyzedreaction in the GPP is provided. The above described kits may comprisean inhibitor of two or more metabolites associated with anenzyme-catalyzed reaction in the GPP. In certain aspects, theinhibitor(s) in the above-described kits can target a metaboliteselected from the group consisting of glutamine, glutamate, aspartate,oxaloacetate, malate, pyruvate, NADH, NADPH, and GSH. Preferably, thekits further comprise instructions for using the kit for treating orpreventing a cancer expressing an oncogenic Kras mutation.

Preferably, in any of the above embodiments, the subject has beenpreviously determined or is simultaneously determined to comprise theoncogenic Kras mutation (e.g., an oncogenic Kras mutation associatedwith cancer). Further the oncogenic Kras mutation can be selected fromthe group consisting of Kras^(G12D), Kras^(G12V), Kras^(G13D),Kras^(G12C), Kras^(Q61R), Kras^(Q61L), Kras^(Q61K), Kras^(G12R), andKras^(G12C). Preferably, the cancer associated with an oncogenic Krasmutation is selected from the group consisting of pancreatic cancer,non-small cell lung cancer, colorectal cancer, and biliary cancer. Mostpreferably, the cancer is pancreatic cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Thematerials, methods, and examples disclosed herein are illustrative onlyand not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present invention. Other features, objects,and advantages of the invention will be apparent from the descriptionand drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A contains a bar graph showing the relative colony number on they-axis for each of the indicated groups of PDAC cultured in 10 cm tissueculture dishes in the presence (+) or absence (−) of glucose (Glc) andglutamine (Gln). Error bars represent standard deviations (s.d.; n=3);**, p<0.01.

FIG. 1B contains line graphs with the relative growth of the indicatedcells types (8988T—upper left quadrant; Panc1—upper right quadrant;PL45—lower left quadrant; MPanc96—lower right quadrant) on the y-axisand the number of days of culture under the indicated conditions on thex-axis. Cells were plated in the complete culture medium (10 mM glucoseand 2 mM Gln) which was replaced the following day with glucose-free(+Gln) or Gln-free medium (+Glc), or with Glc-free and Gln-free medium(Neither), or with medium containing both Glc and Gln (+Glc/Gln). Ineach condition, the medium was supplemented with 10% dialyzed FBS. Errorbars represent s.d. (n=3); **, p<0.01.

FIG. 2 contains a bar graph showing the relative colony number(clonogenic growth) on the y-axis for each of the indicated groups of8988T cells expressing a control shRNA (GFP) or two independent shRNAsto GLS (#1 and #2) and cultured in 10 cm tissue culture dishes. Errorbars represent s.d. (n=3); **, p<0.01.

FIG. 3 is a line graph plotting the relative growth of 8988T cellsexpressing a control shRNA (GFP) or shRNAs to GLS (#1 and #2) on they-axis versus the number of days of culture on the x-axis. Error barsrepresent s.d. (n=3); **, p<0.01.

FIG. 4 is a schematic diagram of Gln metabolism. Abbreviations are asfollows: Gln: glutamine; Glu: glutamate; αKG: α-ketoglutarate; TCAcycle: tricarboxylic acid cycle; NEAA: nonessential amino acid; GLS:glutaminase; GLUD: glutamate dehydrogenase. The dashed-line arrowindicates transport of glutamate in and out of themitochondria/cytoplasm; solid-line arrows indicate an enzymaticreaction, and are drawn from the substrate metabolite to the productmetabolite of the reaction.

FIG. 5 contains a bar graph showing the relative colony number(clonogenic growth) of 8988T cells cultured in 10 cm dishes in thepresence (+) or absence (−) of glucose (Glc), glutamine (Gln),α-ketoglutarate (αKG), and nonessential amino acid (NEAA). Error barsrepresent s.d. (n=3);**, p<0.01.

FIG. 6 contains line graphs with the relative growth of the indicatedcells types (8988T—upper left quadrant; Panc1—upper right quadrant;PL45—lower left quadrant; MPanc96—lower right quadrant) on the y-axisand the number of days of culture under the indicated conditions on thex-axis following glutamine deprivation. Cells were plated in thecomplete culture medium (10 mM glucose and 2 mM glutamine (Gln)) whichwas replaced the following day with glutamine-free medium containing1×NEAA (+NEAA), or 3 mM of dimethyl αKG (+αKG), or the combination(+NEAA/αKG). One group received medium containing glutamine as apositive control (+Gln) and one group was deprived of glutamine as anegative control (none). Error bars represent s.d. (n=3); **, p<0.01.

FIGS. 7A and 7B contain line graphs plotting relative growth of 8988Tcells treated with the indicated concentration of either EGCG(Epigallocatechin Gallate)) (FIG. 7A) or AOA (aminooxyacetate (FIG. 7B)or vehicle and assayed for cell growth. The number of days of culture isshown on the x-axis; error bars represent s.d. (n=3); **, p<0.01.

FIG. 8 contains a bar graph showing the relative colony number(clonogenic growth) of 8988T cells expressing a control shRNA (shGFP) ortwo independent shRNAs to PSAT1 (#1 and #2); GPT2 (#1 and #2); GLUD1 (#1and #2); and GOT1 (#1 and #2) cultured in 10 cm tissue culture dishes.Error bars represent s.d. (n=3); **, p<0.01.

FIG. 9 contains line graphs plotting the relative growth of 8988T cellsexpressing a control shRNA (GFP), or shRNAs to PSAT1 (#1 and #2) (upperleft quadrant); shRNAs to GPT2 (#1 and #2) (upper right quadrant);shRNAs to GLUD1 (#1 and #2) (lower left quadrant), or shRNAs to GOT1 (#1and #2) (lower right quadrant) versus the number of days of culture onthe x-axis; error bars represent s.d. (n=3); **, p<0.01.

FIG. 10 contains a bar graph plotting the relative clonogenic growth of8902 or Miapaca pancreatic cancer cells expressing a control shRNA(shGFP) or shRNAs to GOT1 (#1 and #2) cultured in 10 cm tissue culturedishes. Error bars represent s.d. (n=3); **, p<0.01.

FIG. 11A is a line graph plotting the xenograft growth (expressed astumor volume (mm³)) of 8988T cells expressing a control shRNA (shGFP) orshRNAs targeting GLUD1 (#1 and #2) in mice (n=10). The numbers to rightof graph represent the knockdown efficiency of shRNAs measured beforeinjecting the cells into mice; error bars represent s.e.m. (n=10).

FIG. 11B is a line graph plotting the xenograft growth (expressed astumor volume (mm³)) of 8988T cells expressing a control shRNA (shGFP),shRNAs to GOT1 (#1 and #2), MDH1 (#1 and #2) or ME1 (#1 and #2) in mice;error bars represent s.e.m. (n=10); *, p<0.05; **, p<0.01.

FIG. 12 is a bar graph plotting the relative metabolite abundance of8988T cells grown in U—¹³C Gln upon GOT1 knockdown and presented asthose derived from Gln (U—¹³C) or total metabolite pools (¹²C+U—¹³C)(“total metabolite”). shGFP was used as a control. Abbreviations are asfollows: glutamine (Gln); α-ketoglutarate (αKG), glutamate (Glu);aspartate (Asp); oxaloacetate (OAA); reduced glutathione (GSH); oxidizedglutathione (GSSG). Error bars represent s.d. (n=3); *, p<0.05; **,p<0.01.

FIG. 13 is a bar graph plotting the ratio of reduced to oxidizedglutathione (GSH/GSSG) in 8988T cells expressing a control shRNA (shGFP)or a GOT1 shRNA (#1 or #2) as measured in a biochemical assay. In theshGFP groups, BSO (buthionine sulfoximine) was included as a positivecontrol for GSH depletion. Error bars represent s.d. (n=3); **, p<0.01.

FIGS. 14-17 contain bar graphs plotting the relative ROS (reactiveoxygen species) levels (expressed as DCFDA signal) of 8988T cellsexpressing a control shRNA (shGFP) or a GOT1 shRNA (shGOT1) and culturedunder the indicated conditions: with (+) or without (−) glutamine (Gln),oxaloacetate (OAA), and/or malate, as indicated. Each bar represents themean of three separate experiments; *, p<0.05.

FIG. 18 is a schematic diagram of the pathway by which pyruvate isderived from Gln (in a pathway termed the “glutamine to pyruvate pathway(GPP)”). The dashed-line straight arrow indicates multiple enzymaticreactions; solid-line straight arrow indicates a single enzymaticreaction; curved arrows represent the conversion of additionalsubstrates of the indicated reactions to the listed metabolites.

FIG. 19A is a bar graph plotting the relative metabolite abundance in8988T cells grown in U—¹³C Gln upon ME1 knockdown with shME1 andpresented as those metabolites derived from Gln (U—¹³C) or total pools(¹²C+U—¹³C). shGFP was used as a control. Abbreviations are as follows:glutamine (Gln); α-ketoglutarate (αKG), glutamate (Glu); aspartate(Asp); oxaloacetate (OAA); reduced glutathione (GSH); oxidizedglutathione (GSSG). Error bars represent s.d. (n=3); *, p<0.05; **,p<0.01.

FIG. 19B is a bar graph plotting the NADP1/NADPH ratio in 8988T cellsexpressing a control shRNA (shGFP), or an shRNA to G6PD, IDH1 (G6PD orisocitrate dehydrogenase), GOT1 or ME1; error bars represent s.d. of sixreplicate wells from a representative experiment; **p<0.01.

FIGS. 20A and 20B are bar graphs plotting the mitochondrial NADHINAD+ratio (FIG. 20A) and the oxidative phosphorylation, as measured byoxygen consumption (FIG. 20B) determined in 8988T cells expressing acontrol shRNA (GFP), shRNAs to GOT1 (#1 and #2) or MDH1 (#1) or GLUD1(#1 and #2), as indicated; error bars represent s.d. (n=3); *, p<0.05.

FIG. 20C is a bar graph quantifying the relative clonogenic growth of8988T expressing a control shRNA (shGFP) or shRNAs to GOT2 (#1 and #2);error bars represent s.d. of triplicate wells from a representativeexperiment. **, p<0.01.

FIG. 20D is a bar graph quantifying the fraction of the total metabolitepool that is unlabeled (¹²C), ¹³C-labeled on 2 carbons (²C-¹³C), 3carbons (³C-¹³C) or uniformly labeled (⁴C-¹³C), as determined by Aspisotopomer analysis following GLUD1, GOT1 or GOT2 knockdown, as comparedto shGFP control.

FIG. 20 E contains line graphs plotting the flux of the Gln carbonskeleton into downstream metabolites as a function of time. Reads forion current for [U—¹³C]-labeled metabolites are plotted for cellsexpressing a control shRNA (shGFP) or shRNA to GOT2; error barsrepresent the s.d. of three independently prepared samples. *, p<0.05;**, p<0.01.

FIGS. 21A and 21B contain bar graphs plotting the relative clonogenicgrowth of 8988T cells expressing a control shRNA (shGFP) or shMDH1 (#1or #2) or shME1 (#1 or #2) and cultured in 10 cm tissue culture dishes;error bars represent s.d. (n=3); **, p<0.01.

FIG. 22 is a bar graph plotting the relative colony number (clonogenicgrowth) of 8988T cells plated in complete culture medium (10 mM glucoseand 2 mM Gln) which was replaced the following day with Gln-free mediumsupplemented with the indicated combinations (with (+) or without (−))of nonessential amino acid (NEAA), aspartate (Asp) (2 mM),α-ketoglutarate (αKG) (4 mM) or oxaloacetate (OAA) (4 mM); error barsrepresent s.d. (n=3).

FIG. 23 is a bar graph plotting the relative colony number (clonogenicgrowth) of 8988T cells cultured in 10 cm tissue culture dishes under theindicated conditions: with (+) or without (−) glutamine (Gln) andoxaloacetate (OAA); error bars represent s.d. (n=3); *, p<0.05.

FIG. 24 contains a bar graph plotting the relative colony number(clonogenic growth) of 8988T cells expressing a control shRNA (shGFP) orshRNAs to GLS (#1 and #2) and cultured in complete medium supplementedwith (+) or without (−) oxaloacetate (OAA), as indicated; error barsrepresent s.d. (n=3); **, p<0.01.

FIG. 25 is a bar graph plotting the relative ROS levels in 8988T cellsunder conditions indicated as determined by DCFDA(29,79-dichlorodihydrofluorescein diacetate) staining. DCFDA assay wasperformed 24 h after supplementing Gln-free media with OAA (4 mM). Eachbar represents the mean of three independent experiments with error barsrepresenting the s.d.;*, p<0.05.

FIG. 26 contains a bar graph plotting the relative clonogenic growth ofTu8902, Miapaca2 and Panc1 cells. OAA (4 mM) or GSH (4 mM) was added(indicated by “+”) to media after Gln-withdrawal (indicated by “−”);error bars represent s.d. of triplicate wells from a representativeexperiment. **, p<0.01.

FIG. 27 is a line graph plotting the relative growth of 8988T cellsplated in complete culture media, which was replaced the following daywith Gln-free medium containing dimethyl-malate (4 mM) or OAA (4 mM).Error bars represent s.d. of triplicate wells from a representativeexperiment. **, p<0.01.

FIG. 28 is a line graph plotting the relative growth of 8988T cellsexpressing a control shRNA (shGFP) or shRNAs targeting GOT1, which wereplated in the complete culture media with or without dimethyl-malate (4mM) or OAA (4 mM) and assayed for proliferation; error bars represents.d. of triplicate wells from a representative experiment; **, p<0.01.

FIG. 29 contains a bar graph plotting the relative clonogenic growth of8988T cells under conditions indicated. Cells were plated in completeculture media (10 mM glucose and 2 mM Gln), which was replaced thefollowing day with Gln-free medium supplemented with OAA (4 mM), GSH (4mM) or N-acetylcysteine (NAC) (4 mM); error bars represent s.e.m.(n=10); **, p<0.01.

FIGS. 30A and 30B contain line graphs plotting the relative growth overtime (days) of HPDE (non-transformed human pancreatic ductal epithelialcells), IMR90 (human diploid fibroblasts) and 8988T treated with theindicated concentrations of AOA (FIG. 30A) or EGCG (FIG. 30B) andassayed for proliferation; error bars represent s.d. of triplicate wellsfrom a representative experiment; **, p<0.01.

FIGS. 31A and 31B contain line graphs plotting the relativeproliferation of HPDE (FIG. 31A) and IMR90 (FIG. 31B) cells expressing acontrol shRNA (shGFP) or shRNAs targeting GOT1 (#1 and #2); error barsrepresent s.d. of triplicate wells from a representative experiment. Theinset in each graph is a photograph of a Western blot resultdemonstrating GOT1 knockdown with the shRNA targeting GOT1 in the cells.

FIG. 32A contains a bar graph (left panel) plotting the relativeexpression of GLUD1 and GOT1 as determined by quantitative RT-PCR in8988T cells expressing a control shRNA (shGFP) or two independent shRNAsto Kras (#1 and #2); error bars represent s.d. (n=3); **, p<0.01. Theright panel of the figure shows a Western blot result confirmingknockdown of Kras expression; PActin was used as a loading control.

FIG. 32B is an image of Western blot results showing the protein levelsof Kras, GLUD1, GOT1 and PActin (loading control) in Panc1 cellsexpressing a doxycycline-inducible Kras shRNA at the indicated timepoints (0 and 72 hours (hr)). Error bars represent s.d. (n=3).

FIG. 32C contains bar graphs quantifying relative expression of GLUD1and GOT1 as determined by quantitative RT-PCR in PDAC cell lines (8988T,Tu8902, Panc1, Miapaca2, PL45 and MPanc96) and low passage primary humanPDAC cell lines (#1 and #2) expressing a control shRNA (shGFP) or twoindependent shRNAs to Kras (#1 and #2); error bars represent s.d.; **,p<0.01.

FIG. 32D is a bar graph quantifying the relative expression (relative toKras^(G12D) “On”) of GLUD1 and GOT1 determined by quantitative RT-PCR infive independent orthotopic tumors derived from inducible Kras mice;error bars represent s.d.; **, p<0.01.

FIG. 33 is a bar graph plotting the relative expression of MDH1 or ME1mRNA as determined by quantitative RT-PCR in 8988T cells expressingshRNAs to Kras (#1 or #2) or a control shRNA (shGFP); error barsrepresent s.d. (n=3).

FIG. 34 contains a bar graph plotting the relative colony number(clonogenic growth) of 8988T cells cultured in 10 cm tissue culturedishes and expressing a control shRNA (GFP) or shRNA to Kras (shKras)following treatment with the indicated concentrations of aminooxyacetate(AOA) or Epigallocatechin Gallate (EGCG); error bars represent s.d.(n=3).

FIG. 35 is a bar graph plotting the relative metabolite abundancepresented as those derived from Gln (U—¹³C) or total pools (¹²C+U—¹³C)upon Dox-inducible Kras knockdown in Miapaca pancreatic cancer cells.Abbreviations are as follows: glutamine (Gln); glutamate (Glu);aspartate (Asp); α-ketoglutarate (αKG), oxaloacetate (OAA); error barsrepresent s.d. (n=3); **, p<0.01.

FIG. 36 is a bar graph plotting the relative cell viability of 8988T orPanc1 cells treated with the indicated concentrations of glutaminase(GLS) inhibitors 365 (inactive form), 968 (active form), or BPTES. Errorbars represent s.d. (n=3).

FIGS. 37 and 38 are bar graphs plotting the relative cell viability of8988T cells (FIG. 37) or Panc1 cells (FIG. 38) treated with GLSinhibitors 968 (active form) (10 mM), 365 (inactive form) (50 mM), orBPTES (100 nM) with increasing concentrations of H₂O₂; error barsrepresent s.d. (n=3)); **, p<0.01.

FIG. 39 is a schematic diagram modeling how Gln metabolic reprogrammingin PDAC cells is mediated by oncogenic Kras to maintain cellular redox.The dashed line at the MDH1 step represents shuttling of reducingpotential into the mitochondria. Solid-line arrows indicate enzymaticreactions; abbreviations are as follows: glutamine (Gln), aspartate(Asp), oxaloacetate (OAA), reduced glutathione (GSH), oxidizedglutathione (GSSG), α-ketoglutarate (αKG), glutamate (Glu),tricarboxylic acid cycle (TCA cycle).

DETAILED DESCRIPTION

Various aspects of the invention are described below.

I. Overview

During the process of tumorigenesis, genetic and epigenetic alterationsfine-tune metabolism in cancer cells in a manner that optimizes theirgrowth and survival in the tumor microenvironment. There has been arenewed interest in understanding the altered metabolism in cancer, andhow such dependencies can be targeted for therapeutic gain. However,because normal cells may require the same metabolic pathways as cancercells, achieving a successful therapeutic index remains a majorchallenge to the development of effective cancer therapies that targetmetabolic pathways. Oncogenic Kras reprograms PDAC metabolism to enhanceglucose flux into anabolic pathways. In particular, oncogenic Kraspromotes the diversion of glucose carbons into the non-oxidative arm ofthe pentose phosphate pathway (PPP) without changing glucose fluxthrough the oxidative arm of the PPP. In doing so, oncogenic Krassignaling permits the non-oxidative PPP-mediated generation of ribose(for DNA/RNA biosynthesis) without affecting oxidative PPP-mediatedredox control (NADP+:NADPH balance). This decoupling of ribosebiogenesis from NADPH production suggests that PDAC cells rely on analternative mechanism, independent of glucose metabolism through thePPP, to maintain cellular redox balance.

Recent evidence demonstrates that some cancer cells utilize glutamine(Gln) to support anabolic processes that fuel proliferation. However,the importance of Gln metabolism in pancreatic tumor maintenance is notknown. It is demonstrated in the present Examples that PDAC cells, whichare oncogenic Kras-associated cancer cells, depend on Gln for growth, asPDAC cells were profoundly sensitive to Gln deprivation. Moreover, thepresent Examples demonstrate that PDAC utilizes Gln to generate OAA viaGOT1, and, sequentially, this OAA is converted into malate and thenpyruvate, in a non-canonical pathway of glutamine metabolism, termedherein the “glutamine to pyruvate pathway (GPP).” This series ofreactions results in the transport of NADH into the mitochondria (foroxidative phosphorylation), while simultaneously providing the reducingpower necessary to sustain PDAC growth and survival through reducingequivalents generated by ME1 upon conversion of malate into pyruvate.Importantly, oncogenic Kras appears to support this pathway through theregulation of expression of key metabolic enzymes (GOT1 and GLUD1), andthis reprogramming of Gln metabolism is indispensable for tumormaintenance in PDAC. The presently discovered GPP utilized by PDAC issummarized in the schematic diagram shown in FIG. 18. Moreover, sincePDAC is an oncogenic Kras-associated cancer, other Kras-associatedcancers, e.g., non-small cell lung cancer, colorectal cancer, andbiliary cancer, are also expected to utilize the GPP.

Thus, based at least in part on the above-described discoveries,provided herein are methods for determining the efficacy ofGPP-targeting (e.g., inhibition of an enzyme or metabolite in the GPP)in a subject (e.g., a subject with a cells (e.g., cancer cell)comprising an oncogenic Kras mutation. Also provided are methods fortargeting the GPP, e.g. for the treatment or prevention of cancer, in asubject comprising an oncogenic Kras mutation, the method comprisingadministering to the subject a therapeutically effective amount of acomposition comprising an inhibitor of an enzyme associated with anenzyme-catalyzed reaction in the GPP (e.g., GLS, GOT2, ME1, etc.). Theseembodiments and others are described in detail below.

II. Definitions

As used herein, the term “glutamine to pyruvate pathway (GPP)” means thepresently discovered non-canonical pathway of glutamine metabolismdepicted in FIG. 39, and as described in detail herein. Briefly, the GPPcomprises GOT1, MDH1, and ME1, as well as GLS, GOT2 and other enzymescatalyzing the conversion of glutamate to aspartate, and involves theconversion of glutamine to glutamate, the conversion of glutamate toaspartate (Asp) catalyzed, e.g., by GOT2, the conversion of Asp tooxaloacetate (OAA) catalyzed by GOT1, the conversion of OAA to malatecatalyzed by MDH1 and the conversion of malate to pyruvate catalyzed byME1.

The term “metabolite,” as used herein, means a substrate for or productof an enzymatic reaction of a metabolic pathway, e.g., glutaminemetabolism, as described herein, or a cofactor or agent whose level isregulated by such an enzymatic reaction, and includes reactive oxygenspecies (ROS). Non-limiting examples of such metabolites include, e.g.,NADP+, NADPH, GSSG, GSH, pyruvate, ROS (e.g., hydrogen peroxide, superoxide, hydroxyl radical, hypochlorous acid, nitric oxide, peroxylradical, and singlet oxygen), glutamine, glutamate, aspartate, αKG,NAD+, NADH, oxaloacetate, and malate.

The enzymes and metabolites described herein are also in some contextsreferred to as “markers,” e.g., when their levels or activities aremeasured in order to determine the efficacy of GPP-targeting (e.g., forthe treatment of a cancer associated with an oncogenic Kras mutation).

As used herein, the term “glutamine to pyruvate pathway-targeting” or“GPP-targeting” means a therapy (e.g., treatment of a subject, e.g.,administration of a drug, e.g., an inhibitor or agonist, to the subject)that modulates (i.e., increases or decreases) the expression level oractivity of one or more enzymes or metabolites that are “associated withan enzyme-catalyzed reaction in the GPP” (i.e., an enzyme that itselfcatalyzes a reaction (e.g., GLS, GOT2, GOT1, MDH1, ME1), or thatmodulates the expression or activity of another enzyme that catalyzes areaction in the GPP (e.g., oncogenic Kras, which modulates the activityof GOT1 and modulates the expression of MDH1 and ME1) or an enzyme thatmodulates the level of a metabolite that is a substrate in the GPP; or,i.e., a metabolite that serves as a substrate for or is a product of anenzyme catalyzed reaction in the GPP (e.g., glutamine (Gln), glutamate(Glu), Asp, OAA, malate, αKG, NADH (e.g., mitochondrial or cytoplasmicNADH), NAD+ (e.g., mitochondrial or cytoplasmic NAD+), NADP+, NADPH,cellular reduced glutathione (GSG), oxidized glutathione (GSSG), andpyruvate). GPP-targeting can include a cancer therapy, and preferably,includes a therapy for a cancer associated with an oncogenic Krasmutation, e.g., pancreatic cancer.

As used herein, “targeting an enzyme or metabolite associated with anenzyme-catalyzed reaction in the GPP that is upstream of the malicenzyme (ME1)-catalyzed reaction” means that the therapy (i.e.,GPP-targeting) modulates the expression level or activity of an enzymeor metabolite that is associated with an enzyme-catalyzed reaction inthe GPP before the step in the pathway in which ME1 catalyzes theconversion of malate to pyruvate. Thus, ME1 itself is not a target in atherapy that has a target upstream of ME1, and, e.g., pyruvate, NADP+,NADPH, GSH, GSSG, and ROS which are metabolites that are associated withan enzyme-catalyzed conversion of malate to pyruvate by ME1 are notencompassed by a therapy that has a target upstream of ME1.

As used herein, the terms “Kras-associated cancer” and “cancerassociated with an oncogenic Kras mutation” mean a cancer in which theinitiation and/or maintenance are/is dependent, at least in part, on anactivating mutation in a Kras gene. Typically, an “activating mutation”is one which leads to constitutive activation of the Kras gene andprotein expression of the oncogenic Kras. Non-limiting examples ofactivating mutations in the Kras gene include, e.g., Kras^(G12)),Kras^(G12V), Kras^(G13D), Kras^(G12C), Kras^(Q61R), Kras^(Q61L),Kras^(Q61K), Kras^(G12R), and Kras^(G12C). Non-limiting examples of aKras-associated cancer include human pancreatic ductal adenocarcinoma(PDAC), non-small cell lung cancer, colorectal cancer, and biliarycancer.

As used herein, the term “subject” means any animal, including anymammal, and, in particular, a human. As used herein, a “subjectcomprising an oncogenic Kras mutation” comprises at least one cell thatcontains an oncogenic Kras mutation, as described above. As used herein,a “subject with cancer associated with an oncogenic Kras mutation”comprises at least one cancer cell (e.g., tumor cell) (e.g. pancreaticcancer) that contains an oncogenic Kras mutation, as described above. Incertain embodiments, the subject having such cancer may or may notexhibit other clinical signs of cancer, and may or may not have beendiagnosed with the cancer (e.g. by an attending physician). As usedherein a “subject comprising an oncogenic Kras mutation” comprises atleast one cell comprising the oncogenic Kras mutation; however, thesubject may or may not have been diagnosed with cancer, and may or maynot have cancer.

As used herein, the term “sample” includes any suitable specimenobtained from a subject that includes at least one cell having anoncogenic Kras mutation. Non-limiting examples of suitable specimensinclude, e.g., tumor tissue (e.g., from a biopsy), lymphoid (e.g., lymphnode) tissue, fluid from a cyst, and body fluid such as blood or urine.Where tumor cell containing tissue is used, as appropriate, histologicalsections of tumors or cancer cell-containing tissue, whole or solublefractions of tissue or cell (e.g., cancer cell) lysates, cellsubfractions (e.g., mitochondrial or nuclear subfractions), whole orsoluble fractions of tissue or cell (e.g., cancer cell) subfractionlysates can be analyzed.

As used herein, a “a control level of a marker” is the level of themarker in a sample obtained from the same subject prior to or at thebeginning of the GPP-targeting or from another subject or subjects whois/are known to have a cancer associated with an oncogenic Kras mutation(e.g., KrasG^(G12D)), and is not undergoing or has not undergoneGPP-targeting, and preferably, although not necessarily, any othercancer therapies. In certain embodiments, the control level may be a“predetermined reference level” (i.e., standard) to which the level ofthe marker in the sample from a subject who is undergoing or hasundergone the GPP-targeting is compared. It is understood, that any such“control level” can be a mean level obtained from a plurality of thesubjects referred to above.

As used herein, “treating” or “treatment” of a state, disorder orcondition includes: (1) Preventing or delaying the appearance ofclinical or sub-clinical symptoms of the state, disorder or conditiondeveloping in a mammal that may be afflicted with or predisposed to thestate, disorder or condition but does not yet experience or displayclinical or subclinical symptoms of the state, disorder or condition; or(2) inhibiting the state, disorder or condition, i.e., arresting,reducing or delaying the development of the disease or a relapse thereof(in case of maintenance treatment) or at least one clinical orsub-clinical symptom thereof; or (3) relieving the disease, i.e.,causing regression of the state, disorder or condition or at least oneof its clinical or sub-clinical symptoms. The benefit to a subject to betreated is either statistically significant or at least perceptible tothe patient or to the physician.

As used herein, the term “treating cancer” means causing a partial orcomplete decrease in the rate of growth of a tumor, and/or in the sizeof the tumor and/or in the rate of local or distant tumor metastasis inthe presence of an inhibitor of the invention, and/or any decrease intumor survival.

As used herein, the term “preventing a disease” (e.g., preventing cancerassociated with an oncogenic Kras mutation) in a subject means forexample, to stop the development of one or more symptoms of a disease ina subject before they occur or are detectable, e.g., by the patient orthe patient's doctor. Preferably, the disease (e.g., cancer) does notdevelop at all, i.e., no symptoms of the disease are detectable.However, it can also result in delaying or slowing of the development ofone or more symptoms of the disease. Alternatively, or in addition, itcan result in the decreasing of the severity of one or more subsequentlydeveloped symptoms.

As used herein “combination therapy” means the treatment of a subject inneed of treatment with a certain composition or drug in which thesubject is treated or given one or more other compositions or drugs forthe disease in conjunction with the first and/or in conjunction with oneor more other therapies, such as, e.g., a cancer therapy such aschemotherapy, radiation therapy, and/or surgery. Such combinationtherapy can be sequential therapy wherein the patient is treated firstwith one treatment modality (e.g., drug or therapy), and then the other(e.g., drug or therapy), and so on, or all drugs and/or therapies can beadministered simultaneously. In either case, these drugs and/ortherapies are said to be “coadministered.” It is to be understood that“coadministered” does not necessarily mean that the drugs and/ortherapies are administered in a combined form (i.e., they may beadministered separately or together to the same or different sites atthe same or different times).

The term “pharmaceutically acceptable derivative” as used herein meansany pharmaceutically acceptable salt, solvate or prodrug, e.g., ester,of a compound of the invention, which upon administration to therecipient is capable of providing (directly or indirectly) a compound ofthe invention, or an active metabolite or residue thereof. Suchderivatives are recognizable to those skilled in the art, without undueexperimentation. Nevertheless, reference is made to the teaching ofBurger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1:Principles and Practice, which is incorporated herein by reference tothe extent of teaching such derivatives. Preferred pharmaceuticallyacceptable derivatives are salts, solvates, esters, carbamates, andphosphate esters. Particularly preferred pharmaceutically acceptablederivatives are salts, solvates, and esters. Most preferredpharmaceutically acceptable derivatives are salts and esters.

As used herein the terms “therapeutically effective” and “effectiveamount”, used interchangeably, applied to a dose or amount refers to aquantity of a composition, compound or pharmaceutical formulation thatis sufficient to result in a desired activity upon administration to ananimal in need thereof. Within the context of the present invention, theterm “therapeutically effective” refers to that quantity of acomposition, compound or pharmaceutical formulation that is sufficientto reduce or eliminate at least one symptom of a disease or conditionspecified herein. When a combination of active ingredients isadministered, the effective amount of the combination may or may notinclude amounts of each ingredient that would have been effective ifadministered individually. The dosage of the therapeutic formulationwill vary, depending upon the nature of the disease or condition, thepatient's medical history, the frequency of administration, the mannerof administration, the clearance of the agent from the host, and thelike. The initial dose may be larger, followed by smaller maintenancedoses. The dose may be administered, e.g., weekly, biweekly, daily,semi-weekly, etc., to maintain an effective dosage level.

Therapeutically effective dosages can be determined stepwise bycombinations of approaches such as (i) characterization of effectivedoses of the composition or compound in in vitro cell culture assaysusing tumor cell growth and/or survival as a readout followed by (ii)characterization in animal studies using tumor growth inhibition and/oranimal survival as a readout, followed by (iii) characterization inhuman trials using enhanced tumor growth inhibition and/or enhancedcancer survival rates as a readout.

The term “nucleic acid hybridization” refers to the pairing ofcomplementary strands of nucleic acids. The mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleobases) of the strands of nucleic acids. Forexample, adenine and thymine are complementary nucleobases that pairthrough the formation of hydrogen bonds. Hybridization can occur undervarying circumstances. Nucleic acid molecules are “hybridizable” to eachother when at least one strand of one nucleic acid molecule can formhydrogen bonds with the complementary bases of another nucleic acidmolecule under defined stringency conditions. Stringency ofhybridization is determined, e.g., by (i) the temperature at whichhybridization and/or washing is performed, and (ii) the ionic strengthand (iii) concentration of denaturants such as formamide of thehybridization and washing solutions, as well as other parameters.Hybridization requires that the two strands contain substantiallycomplementary sequences. Depending on the stringency of hybridization,however, some degree of mismatches may be tolerated. Under “lowstringency” conditions, a greater percentage of mismatches are tolerable(i.e., will not prevent formation of an anti-parallel hybrid). SeeMolecular Biology of the Cell, Alberts et al., 3rd ed., New York andLondon: Garland Publ., 1994, Ch. 7.

Typically, hybridization of two strands at high stringency requires thatthe sequences exhibit a high degree of complementarity over an extendedportion of their length. Examples of high stringency conditions include:hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at65° C., followed by washing in 0.1×SSC/0.1% SDS (where 1×SSC is 0.15 MNaCl, 0.15 M Na citrate) at 68° C. or for oligonucleotide (oligo)inhibitors washing in 6×SSC/0.5% sodium pyrophosphate at about 37° C.(for 14 nucleotide-long oligos), at about 48° C. (for about 17nucleotide-long oligos), at about 55° C. (for 20 nucleotide-longoligos), and at about 60° C. (for 23 nucleotide-long oligos).

Conditions of intermediate or moderate stringency (such as, for example,an aqueous solution of 2×SSC at 65° C.; alternatively, for example,hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at65° C. followed by washing in 0.2×SSC/0.1% SDS at 42° C.) and lowstringency (such as, for example, an aqueous solution of 2×SSC at 55°C.), require correspondingly less overall complementarity forhybridization to occur between two sequences. Specific temperature andsalt conditions for any given stringency hybridization reaction dependon the concentration of the target DNA or RNA molecule and length andbase composition of the probe, and are normally determined empiricallyin preliminary experiments, which are routine (see Southern, J. Mol.Biol. 1975; 98:503; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 2, ch. 9.50, CSH Laboratory Press, 1989; Ausubelet al. (eds.), 1989, Current Protocols in Molecular Biology, Vol. 1,Green Publishing Associates, Inc., and John Wiley & Sons, Inc., NewYork, at p. 2.10.3). An extensive guide to the hybridization of nucleicacids is found in, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I, chapt 2, “Overview of principles of hybridization and thestrategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).

As used herein, the term “standard hybridization conditions” refers tohybridization conditions that allow hybridization of two nucleotidemolecules having at least 50% sequence identity. According to a specificembodiment, hybridization conditions of higher stringency may be used toallow hybridization of only sequences having at least 75% sequenceidentity, at least 80% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 99% sequenceidentity.

As used herein, the phrase “under hybridization conditions” meansconditions under conditions that facilitate specific hybridization of asubset of capture oligonucleotides to complementary sequences present inthe cDNA or cRNA. The terms “hybridizing specifically to” and “specifichybridization” and “selectively hybridize to,” as used herein refer tothe binding, duplexing, or hybridizing of a nucleic acid moleculepreferentially to a particular nucleotide sequence under at leastmoderately stringent conditions, and preferably, highly stringentconditions, as discussed above.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification.

As used herein, the term “nucleic acid” or “oligonucleotide” refers to adeoxyribonucleotide or ribonucleotide in either single- ordouble-stranded form. The term also encompasses nucleic-acid-likestructures with synthetic backbones. DNA backbone analogues provided bythe invention include phosphodiester, phosphorothioate,phosphorodithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino),3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs);see Oligonucleotides and Analogues, a Practical Approach, edited by F.Eckstein, IRL Press at Oxford University Press (1991); AntisenseStrategies, Annals of the New York Academy of Sciences, Volume 600, Eds.Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem.36:1923-1937; Antisense Research and Applications (1993, CRC Press).PNAs contain non-ionic backbones, such as N-(2-aminoethyl)glycine units.Phosphorothioate linkages are described in WO 97/03211; WO 96/39154;Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other syntheticbackbones encompassed by the term include methyl-phosphonate linkages oralternating methylphosphonate and phosphodiester linkages(Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonatelinkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156). Theterm nucleic acid is used interchangeably with cDNA, cRNA, mRNA,oligonucleotide, probe and amplification product.

Some of the abbreviations used in the present disclosure are as follows:Gln: glutamine; Glu: glutamate, Glc: glucose; Asp: aspartate; OAA:oxaloacetate; αKG: α-ketoglutarate; ROS: reactive oxygen species; NAD+:nicotinamide adenine dinucleotide; NADH: reduced nicotinamide adeninedinucleotide; NADP+: nicotinamide adenine dinucleotide phosphate; NADPH:reduced nicotinamide adenine dinucleotide phosphate; GSSG: glutathionedisulfide; GSH: reduced glutathione; GLS: glutaminase; GOT:glutamic-oxaloacetic transaminase; MDH: malate dehydrogenase; ME: malicenzyme.

III. Oncogenic Kras

The human Kras gene sequence has two preferred transcript variants,having the nucleic acid sequences given in GenBank Accession Nos.NM_(—)033360 (transcript variant a) (SEQ ID NO: 1) and NM_(—)004985.3(transcript variant b) (SEQ ID NO: 2). The human Kras protein sequencehas two preferred variants, having the amino acid sequence given inGenBank Accession Nos. NP_(—)203524 (isoform a) (SEQ ID NO: 3) andNP_(—)004976.2 (isoform b) (SEQ ID NO: 4). The numbering of Kras aminoacid mutations (e.g., Kras^(G12D), Kras^(G61R), etc., corresponds toeither of the above-given amino acid sequences.

Oncogenic Kras mutations associated with cancer include, withoutlimitation, Kras^(G12D), Kras^(G12V), Kras^(G13D), Kras^(G12C),Kras_(Q61R), Kras^(Q61L), Kras^(Q61K), Kras^(G12R), and Kras^(G12C). Theskilled artisan will understand that a Kras gene comprising a differentKras mutation than one of those above and/or combinations of the aboveand/or other Kras mutations that lead to and/or do not preventactivation, and preferably constitutive activation of Kras, is also anoncogenic Kras encompassed by the present invention. In a preferredembodiment, an oncogenic Kras is Kras^(G12D).

The presence of an oncogenic Kras mutation in a sample, e.g., from acell, tumor biopsy, or other DNA, RNA or protein-containing sample canbe determined at the genomic, RNA or protein level according to anysuitable method known in the art.

For example, Southern blotting can be used to determine the presence ofan oncogenic Kras mutation in a genome. Methods for Southern blottingare known to those of skill in the art (see,e.g., Current Protocols inMolecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York, 1995, or Sambrook et al., MolecularCloning: A Laboratory Manual, 2d Ed. vol. 1-3, Cold Spring Harbor Press,NY, 1989). In such an assay, the genomic DNA (typically fragmented andseparated on an electrophoretic gel) is hybridized to a probe specificfor the target region. Comparison of the intensity of the hybridizationsignal from the probe for the target region with control probe signalfrom analysis of normal genomic DNA (e.g., genomic DNA from the same orrelated cell, tissue, organ, etc.) provides an estimate of the relativecopy number of the target nucleic acid.

Amplification-based assays, such as PCR, can also be used to determinethe presence of an oncogenic Kras mutation in a genome, as well as themRNA expression of an oncogenic Kras in an RNA-containing sample.Detailed protocols for quantitative PCR are provided, for example, inInnis et al. (1990) PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc. N.Y. Real-time PCR can also be used (see, e.g.,Gibson et al., Genome Research 6:995-1001, 1996; Heid et al., GenomeResearch 6:986-994, 1996). Real-time PCR evaluates the level of PCRproduct accumulation during amplification. Total genomic DNA (or RNA) isisolated from a sample. Real-time PCR can be performed, for example,using a Perkin Elmer/Applied Biosystems (Foster City, Calif.) 7700 Prisminstrument. Matching primers and fluorescent probes can be designed forgenes of interest using, for example, the primer express programprovided by Perkin Elmer/Applied Biosystems (Foster City, Calif.).Optimal concentrations of primers and probes can be initially determinedby those of ordinary skill in the art, and control (for example,beta-actin) primers and probes may be obtained commercially from, forexample, Perkin Elmer/Applied Biosystems (Foster City, Calif.). Toquantitate the amount of the specific nucleic acid of interest in asample, a standard curve is generated using a control. Standard curvesmay be generated using the Ct values determined in the real-time PCR,which are related to the initial concentration of the nucleic acid ofinterest used in the assay. Standard dilutions ranging from 10-10⁶copies of the gene of interest are generally sufficient. In addition, astandard curve is generated for the control sequence. This permitsstandardization of initial content of the nucleic acid of interest in atissue sample to the amount of control for comparison purposes. Methodsof real-time quantitative PCR (“QPCR”) using TaqMan probes are wellknown in the art. Detailed protocols for QPCR are provided, for example,for RNA in: Gibson et al., 1996, A novel method for real timequantitative RT-PCR. Genome Res., 10:995-1001; and for DNA in: Heid etal., 1996, Real time quantitative PCR. Genome Res., 10:986-994.

Other suitable amplification methods include, but are not limited toligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4:560,Landegren et al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89:117), transcription amplification (Kwoh et al. (1989) Proc.Natl. Acad. Sci. USA 86:1173), self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87:1874), dot PCR, andlinker adapter PCR, etc. In another embodiment, DNA sequencing may beused to determine the presence of an oncogenic Kras mutation in agenome. Methods for DNA sequencing are known to those of skill in theart.

IV. Enzymes Associated with the GPP

As described above, and as shown in FIG. 39, in addition to Kras, theenzymes GLS, GOT1, GOT2, MDH1, and ME1 are presently demonstrated to beassociated with an enzyme-catalyzed reaction in the GPP. Specifically,the GPP is shown to involve the conversion of Gln to Glu, catalyzed byGLS; the conversion of Glu to Asp, catalyzed, e.g., by GOT2; theconversion of Asp to OAA, catalyzed by GOT1; the conversion of OAA tomalate, catalyzed by MDH1; and the conversion of malate to pyruvate,catalyzed by ME1. Furthermore, the reaction catalyzed by GOT1 alsoreduces αKG to Glu, the reaction catalyzed by MDH1 also oxidizes NADH toNAD+, which is then shuttled to the mitochondria and reduced to NADH,and the reaction catalyzed by ME1 also reduces NADP+ to NADPH, whichfurther results in the reduction of GSSH to GSH and the reduction inlevels of ROS. It will be understood that Gls, GOT2, other enzymesinvolved in conversion of glutamate to aspartate, GOT1, MDH1, and ME1are enzymes in the GPP and that all the enzymes in the GPP and, forexample, Kras and GLUD1 are enzymes associated with the GPP.

The nucleic acid and amino acid sequences for the enzymes GLS, GOT1,GOT2, MDH1, and ME1 presently discovered to be associated with the GPPare known and have been described. GenBank® Accession Nos. of exemplarynucleic acid and amino acid sequences for the human enzymes are providedin Table 1, below.

TABLE 1 Exemplary GenBank ® Accession Numbers for Enzymes Associatedwith GPP Nucleic Acid SEQ Corresponding Amino Acid SEQ GenBank ® IDPolypeptide GenBank ® ID Gene Name Accession No. NO Name Accession No.NO glutaminase (GLS), NM_001256310 5 glutaminase NP_001243239 6 nucleargene kidney isoform, encoding mitochondrial mitochondrial isoform 2(GLS) protein, transcript variant 2 glutamic-oxaloacetic NM_002080 7aspartate NP_002071 8 transaminase 2, aminotransferase, mitochondrialmitochondrial (aspartate precursor aminotransferase 2) (GOT2) (GOT2),nuclear gene encoding mitochondrial protein glutamic-oxaloaceticNM_002079 9 aspartate NP_002070 10 transaminase 1, aminotransferase,soluble (aspartate cytoplasmic aminotransferase 1) (GOT1) (GOT1) malateNM_001199111 11 malate NP_001186040 12 dehydrogenase 1, dehydrogenase,NAD (soluble) cytoplasmic (MDH1), transcript isoform 1 variant 1 (MDH1)malate NM_005917 13 malate NP_005908 14 dehydrogenase 1, dehydrogenase,NAD (soluble) cytoplasmic (MDH1), transcript (MDH1), variant 2 isoform 2malate NM_001199112 15 malate NP_001186041 16 dehydrogenase 1,dehydrogenase, NAD (soluble) cytoplasmic (MDH1), transcript (MDH1),variant 3 isoform 3 malic enzyme 1, NM_002395 17 NADP-dependentNP_002386 18 NADP(+)-dependent, malic enzyme cytosolic (ME1)

In certain embodiments, it is desirable to determine (e.g., assay,measure, approximate) the level (e.g., expression or activity) of anenzyme associated with an enzyme-catalyzed reaction in the GPP. Theexpression level of such an enzyme may be determined according to anysuitable method known in the art. A non-limiting example of such amethod includes PCR, e.g., real-time quantitative PCR (QPCR), asdescribed in detail above, which measures the expression level of themRNA encoding the polypeptide. mRNA expression can also be determinedusing microarray (transcriptomic analysis), methods for which are wellknown in the art (see, e.g., Watson et al. Curr Opin Biotechnol (1998)9: 609-14). For example, mRNA expression profiling can be performed toidentify differentially expressed genes, wherein the raw intensitiesdetermined by microarray are log 2-transformed and quantile normalizedand gene set enrichment analysis (GSEA) is performed according, e.g., toSubramanian et al. (2005) Proc Natl Acad Sci USA 102:15545-15550).

Other examples of suitable methods include Western blot, ELISA and/orimmunohistochemistry, which can be used to measure protein expressionlevel. Such methods are well known in the art.

Methods for assaying the activity of an enzyme of the present inventioninclude functional in vitro assays and are well known in the art. Forexample, and without limitation, the activity of virtually any enzymecan be traced using isotopically labeled molecules and standards (e.g.by MS, HPLC, NMR). More straightforward methods rely on coupling theactivity of a desired enzyme to those with a readily observable readout(like NAD/NADH, NADP+/NADPH, ROS (e.g., total ROS), GSSG/GSH (e.g.,levels of GSH), etc.). Examples of such assays are described, forexample, in Harris and Keshwani (2009), Methods in Enzymology: Guide toProtein Purification, 2nd Edition; 463:57-71; Rossomando, E. F. (1990)Methods in Enzymology: Guide to Protein Purification, 2nd Edition;182:38-49; Crutchfield et al. (2010) Methods in Enzymology; Guide toProtein Purification, 2nd Edition; 470:393-426; Befroy et al. (2009)Methods in Enzymology; Guide to Protein Purification, 2nd Edition:457:373-393; and Bartlett and Causey (1988) Methods in Enzymology: Guideto Protein Purification, 2nd Edition: 166:79-92.

V. Metabolites of the Invention

Non-limiting examples of metabolites associated with an enzyme-catalyzedreaction in the GPP include, e.g., NADP+, NADPH, GSSG, GSH, pyruvate,ROS (e.g., hydrogen peroxide, super oxide, hydroxyl radical,hypochlorous acid, nitric oxide, peroxyl radical, and singlet oxygen),glutamine, glutamate, aspartate, αKG, NAD+, NADH, oxaloacetate, andmalate. Such metabolites are described, e.g., in Berg, J. M. et al.Biochemistry (Textbook) ISBN-10: 0716787245|ISBN-13:978-0716787242|Publication Date: May 19, 2006|Edition: Sixth Edition.

In certain embodiments, it is desirable to measure the levels of one ormore such metabolites in a sample. Metabolite levels may be measuredaccording to any suitable method known in the art. For example,metabolite levels may be measured using targeted liquid-chromatographymass spectrometry (LC/MS/MS). For example, for metabolite collectionfrom cultured cells (at, e.g., ˜70% confluence) is fully aspirated and 4mL of 80% (v/v) methanol is added at dry ice temperatures. Cells and themetabolite-containing supernatants are collected into conical tubes.Insoluble material in lysates are centrifuged at 2,000×g for 15 min, andthe resulting supernatant is evaporated using a refrigerated speed vac.Subsequent metabolite analysis is performed as described before (see,Locasale et al. (2011) Nature Genetics; 43:869-874). Peak areas from thetotal ion current for each metabolite multiple reaction monitoring (MRM)transition are integrated using MultiQuant v1.1 software (AppliedBiosystems). Data analysis was performed in Cluster3.0 and TreeViewer.Such methods are also described in detail in U.S. provisionalapplication No. 61/578,116. The skilled artisan will appreciate thatnuclear magnetic resonance (NMR) can also be used to measure the levelmetabolites as well. Such methods are well known in the art and aredescribed in detail, e.g., in Wishart D S. (2011) Bioanalysis; August;3(15): 1769-82.

ROS can be measured as described in the Examples, e.g., using2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Invitrogen).Oxidation of DCFDA to the highly fluorescent 2′,7′-dichloro-fluorescein(DCF) is detectable (e.g., by flow cytometry) and is proportionate toROS generation.

The skilled artisan will understand, however, that other methods areknown in the art and may be used for measuring the levels of metabolitesin a sample.

VI. Methods of Treatment and Prevention

Methods for treating or preventing an oncogenic Kras-associated cancerin a subject comprising targeting the GPP are provided. A subjectundergoing GPP-targeting may be a subject with a cancer associated withan oncogenic Kras mutation (e.g., pancreatic cancer, non-small cell lungcancer, colorectal cancer, biliary cancer). However, in otherembodiments, the subject undergoing GPP-targeting may not have beendiagnosed with cancer and may or may not have cancer. Such a subject mayhave undergone or may be undergoing GPP-targeting for another reason orother reasons, such as, but not limited to, for the prevention of acancer associated with an oncogenic Kras mtuations. Such patient mayhave been, e.g., determined, e.g., by the subject's physician, tocomprise an oncogenic Kras mutation (e.g., Kras^(G12D), Kras^(G12V),Kras^(G13D), Kras^(G12C), Kras^(Q61R), Kras^(Q61L), Kras^(Q61K),Kras^(G2R), or Kras^(G12C)), and, as such, e.g., at risk of developing acancer associated with the Kras mutation. While not intending to bebound by any one particular theory or mechanism, as demonstrated herein,oncogenic Kras increases the expression of GOT1, and decreases theexpression of GLUD1, thereby driving the non-canonical pathway ofglutamine metabolism described herein (i.e., the GPP); thus, a personhaving an oncogenic Kras mutation, but not necessarily having beendiagnosed with cancer, can benefit from receiving GPP-targeting, e.g.,as a prophylactic treatment (e.g., delaying or preventing the onset ofcancer).

Cancers associated with an oncogenic Kras mutation that may be treatedor prevented according to the present methods, include, e.g., pancreaticcancer, non-small cell lung cancer, colorectal cancer, and biliarycancer. However, the skilled artisan will understand that any cancerthat is associated with an oncogenic Kras mutation is encompassed by thepresent methods and kits pertaining thereto.

In certain embodiments, the method for treatment or prevention comprisesadministering to a subject comprising an oncogenic Kras mutation (e.g.,a subject with an oncogenic Kras-associated cancer or at risk ofdeveloping such cancer (e.g., a subject comprising an oncogenic Krasmutation)) a therapeutically effective amount of a compositioncomprising an inhibitor that targets at least one enzyme or metaboliteassociated with an enzyme-catalyzed reaction in the GPP.

In other embodiments, the treatment method can comprise inhibiting aplurality (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more,7 or more, 8 or more, 9 or more, etc.) enzymes and/or metabolitesassociated with an enzyme-catalyzed reaction in the GPP (e.g., in acombination therapy).

The enzymes and metabolites that can be inhibited, or in certain cases,treated with an agonist to increase activity or expression level,include, but are not limited to, the enzymes GLS, GOT2, GOT1, MDH1, ME1,Kras, and GLUD1, and the metabolites NADP+, NADPH, GSSG, GSH, pyruvate,glutamine, glutamate, aspartate, αKG, NAD+, NADH, oxaloacetate, andmalate. Further, ROS levels may be increased, e.g., by administration ofinhibitors of anti-oxidants. Non-limiting examples of anti-oxidantinhibitors include, e.g., aminotriazole, chlorodonitrobenzene,mercaptosuccinate, and gemcitabine.

Thus, in certain embodiments, an inhibitor or any combination of 2 ormore inhibitors of the above-described enzymes and metabolitesassociated with an enzyme-catalyzed reaction in the GPP can beadministered in a combination therapy to a subject comprising anoncogenic Kras mutation in order to target the GPP (e.g., for thetreatment of cancer).

In a preferred embodiment, the method of treatment comprisesadministering to a subject comprising an oncogenic Kras mutation aninhibitor of the enzyme ME1. In other embodiments, an inhibitor of ME1is administered in a combination therapy with at least one additionalinhibitor, or in certain cases, agonist (e.g., of GLUD1), of an enzymeor metabolite associated with an enzyme-catalyzed reaction in the GPP.For example an inhibitor of ME1 can be administered to a subjectcomprising an oncogenic Kras mutation in a combination therapy with atleast one inhibitor of an enzyme selected from GLS, GOT2, GOT1, MDH1,and Kras, and/or an agonist of GLUD1, and/or an inhibitor of at leastone metabolite selected from NADP+, NADPH, GSSG, GSH, pyruvate,glutamine, glutamate, aspartate, αKG NAD+, NADH, oxaloacetate, andmalate.

In another preferred embodiment, the method of treatment comprisesadministering to a subject comprising an oncogenic Kras mutation aninhibitor of the enzyme GLS. In other embodiments, an inhibitor of GLSis administered in a combination therapy with at least one additionalinhibitor, or in certain cases, agonist (e.g., of GLUD1), of an enzymeor metabolite associated with an enzyme-catalyzed reaction in the GPP.For example an inhibitor of GLS can be administered to a subjectcomprising an oncogenic Kras mutation in a combination therapy with atleast one inhibitor of an enzyme selected from GOT2, GOT1, MDH1, ME1,Kras, and/or an agonist of GLUD1, and/or an inhibitor of at least onemetabolite selected from NADP+, NADPH, GSSG, GSH, pyruvate, glutamine,glutamate, aspartate, αKG, NAD+, NADH, oxaloacetate, and malate.

In another preferred embodiment, the method of treatment comprisesadministering to a subject comprising an oncogenic Kras mutation aninhibitor of the enzyme GOT2. In other embodiments, an inhibitor of GOT2is administered in a combination therapy with at least one additionalinhibitor, or in certain cases, agonist (e.g., of GLUD1), of an enzymeor metabolite associated with an enzyme-catalyzed reaction in the GPP.For example an inhibitor of GOT2 can be administered to a subjectcomprising an oncogenic Kras mutation in a combination therapy with atleast one inhibitor of an enzyme selected from GLS, GOT1, MDH1, ME1,Kras, and/or an agonist of GLUD1 and/or an inhibitor of at least onemetabolite selected from NADP+, NADPH, GSSG, GSH, pyruvate, glutamine,glutamate, aspartate, αKG, NAD+, NADH, oxaloacetate, and malate.

Non-limiting examples of inhibitors are selected from the groupconsisting of anti-sense oligonucleotides, small hairpin RNA (shRNA),small inhibiting RNA (siRNA), intrabodies, aptamers, and small molecule,as described in detail in Section VIII, below.

The skilled artisan will appreciate that other combinations ofinhibitors and/or agonists are possible, so long as the combinationresults in the inhibition of the GPP. Methods for determining theefficacy of GPP-targeting (e.g., inhibition) are described in detail inthe following section.

The skilled artisan will also appreciate that the GPP-targetingdescribed herein, e.g. for the treatment of a cancer associated with anoncogenic Kras mutation, may also be administered in a combinationtherapy with other treatments, e.g. other cancer therapies. Non-limitingexamples of such cancer therapies include chemotherapy, radiationtherapy, antiangiogenic therapy, surgery, and combinations thereof.

In a preferred embodiment, GPP-targeting is administered to a subjectwith a cancer associated with an oncogenic Kras mutation in acombination therapy with a treatment that increases ROS. While notintending to be limited to any one particular theory or mechanism ofaction, the present Examples demonstrate that PDAC drive glutamine intothe GPP, thereby reducing the levels of ROS; accordingly, increasing ROSlevels can be beneficial for reducing cancer cell growth. Moreover,certain cancer therapies are known to increase ROS levels, such as, butnot limited to, radiation therapy, as well as autophagy inhibitors suchas hydroxychloroquine and chloroquine, and anti-oxidant inhibitors suchas aminotriazole, chlorodonitrobenzene, mercaptosuccinate, andgemcitabine. Thus, in certain embodiments, the GPP-targeting (e.g.,administration of an inhibitor, or in certain cases, agonist, of atleast one enzyme or metabolite associated with an enzyme-catalyzedreaction in the GPP) in a subject is administered in a combinationtherapy with a radiation therapy, and/or one or more autophagyinhibitors (e.g., hydroxychloroquine, chloroquine), and/or one or moreanti-oxidant inhibitors (e.g., aminotriazole, chlorodonitrobenzene,mercaptosuccinate, and gemcitabine).

Chemotherapeutic agents, include for example: taxanes such as taxol,taxotere or their analogues; alkylating agents such as cyclophosphamide,isosfamide, melphalan, hexamethylmelamine, thiotepa or dacarbazine;antimetabolites such as pyrimidine analogues, for instance5-fluorouracil, cytarabine, capecitabine, and gemcitabine or itsanalogues such as 2-fluorodeoxycytidine; folic acid analogues such asmethotrexate, idatrexate or trimetrexate; spindle poisons includingvinca alkaloids such as vinblastine, vincristine, vinorelbine andvindesine, or their synthetic analogues such as navelbine, orestramustine and a taxoid; platinum compounds such as cisplatin;epipodophyllotoxins such as etoposide or teniposide; antibiotics such asdaunorubicin, doxorubicin, bleomycin or mitomycin, enzymes such asL-asparaginase, topoisomerase inhibitors such as topotecan orpyridobenzoindole derivatives; and various agents such as procarbazine,mitoxantrone, and biological response modifiers or growth factorinhibitors such as interferons or interleukins. Other chemotherapeuticagents include, though are not limited to, a p38/JAK kinase inhibitor,e.g., SB203580; a phospatidyl inositol-3 kinase (PI3K) inhibitor, e.g.,LY294002; a MAPK inhibitor, e.g. PD98059; a JAK inhibitor, e.g., AG490;preferred chemotherapeutics such as UCN-01, NCS, mitomycin C (MMC), NCS,and anisomycin; taxoids in addition to those describe above (e.g., asdisclosed in U.S. Pat. Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957;5,292,921; 5,438,072; 5,587,493; European Patent No. 0 253 738; and PCTPublication Nos. WO 91/17976, WO 93/00928, WO 93/00929, and WO 96/01815.In other embodiments, a cancer therapy can include but is not limited toadministration of cytokines and growth factors such as interferon(IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta, and/or similarcytokines, or an antagonist of a tumor growth factor (e.g., TGF-β andIL-10). Antiangiogenic agents, include, e.g., endostatin, angiostatin,TNP-470, Caplostatin (Stachi-Fainaro et al., Cancer Cell 7(3), 251(2005)). Drugs that interfere with intracellular protein synthesis canalso be used in the methods of the present invention; such drugs areknown to those skilled in the art and include puromycin, cycloheximide,and ribonuclease.

For radiation therapy, common sources of radiation used for cancertreatment include, but are not limited to, high-energy photons that comefrom radioactive sources such as cobalt, cesium, iodine, palladium, or alinear accelerator, proton beams; neutron beams (often used for cancersof the head, neck, and prostate and for inoperable tumors).

It is well known that radioisotopes, drugs, and toxins can be conjugatedto antibodies or antibody fragments which specifically bind to markerswhich are produced by or associated with cancer cells, and that suchantibody conjugates can be used to target the radioisotopes, drugs ortoxins to tumor sites to enhance their therapeutic efficacy and minimizeside effects. Examples of these agents and methods are reviewed inWawrzynczak and Thorpe (in Introduction to the Cellular and MolecularBiology of Cancer, L. M. Franks and N. M. Teich, eds, Chapter 18, pp.378-410, Oxford University Press. Oxford, 1986), in Immunoconjugates:Antibody Conjugates in Radioimaging and Therapy of Cancer (C. W. Vogel,ed., 3-300, Oxford University Press, N.Y., 1987), in Dillman, R. O. (CRCCritical Reviews in Oncology/Hematology 1:357, CRC Press, Inc., 1984),in Pastan et al. (Cell 47:641, 1986) in Vitetta et al. (Science238:1098-1104, 1987) and in Brady et al. (Int. J. Rad. Oncol. Biol.Phys. 13:1535-1544, 1987). Other examples of the use of immunoconjugatesfor cancer and other forms of therapy have been disclosed, inter alia,in U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744,4,460,459, 4,460,561 4,624,846, 4,818,709, 4,046,722, 4,671,958,4,046,784, 5,332,567, 5,443,953, 5,541,297, 5,601,825, 5,637,288,5,677,427, 5,686,578, 5,698,178, 5,789,554, 5,922,302, 6,187,287, and6,319,500.

Such inhibitors include antisense oligonucleotides (e.g., RNAinterfering molecules such as siRNA and shRNA), aptamers, ribozymes, andsmall molecules, including certain chemotherapeutic agents. Non-limitingexamples of such inhibitors include AZD8330, which is a MEK inhibitor,BKMI20, which is a PI3K inhibitor, and GSK1120212, which is also a MEKinhibitor. The skilled artisan will appreciate that any suitableinhibitor of one or more of the above polypeptides is encompassed by thepresent invention.

VII. Methods for Determining Efficacy of GPP Targeting

Provided herein are methods for determining the efficacy ofGPP-targeting in a subject. As described above, GPP-targeting may becarried out for any number of reasons, although preferably,GPP-targeting is administered to a subject for the treatment orprevention of a cancer associated with an oncogenic Kras mutation. Thus,in preferred embodiments, GPP targeting is administered to a subject whohas been determined to comprise an oncogenic Kras mutation (e.g., as aprophylactic treatment to delay and/or prevent the onset of or to treata cancer associated with the oncogenic Kras mutation). A subject may bepreviously determined or simultaneously determined to comprise anoncogenic Kras mutation (e.g. comprise cells (e.g., cancer cells)expressing an oncogenic Kras mutation).

The presently provided methods for determining the efficacy ofGPP-targeting in a subject typically involve determining (e.g.,measuring, assaying, estimating) the level (e.g., expression and/oractivity) of one or more markers (e.g., enzymes and/or metabolites)associated with an enzyme-catalyzed reaction in the GPP. In a preferredembodiment, the one or more markers measured are selected from themarkers that are upstream of ME1-catalyzed reaction in the GPP. Whilenot intending to be bound by any one particular theory or mechanism ofaction, the present methods are based, at least in part, on thediscovery of a novel pathway utilized by cancer cells that areassociated with an oncogenic Kras mutation which results in the shuntingof glutamine into a non-canonical metabolic pathway (i.e., the GPP) thatresults in, e.g., increased levels of NADPH, GSH and decreased ROSlevels. Thus, inhibition of the GPP can be read out by determiningwhether the level of one or more of the “end products” of the pathway(e.g., NADPH, GSH, ROS) have been altered. Of course, the skilledartisan will also appreciate that the level of intermediates in thepathway, e.g., markers that are upstream of NADPH, GSH, and ROS (e.g.,aspartate, OAA, malate, NAD+, NADH, etc.) can, alternatively, oradditionally, be determined in order to determine the efficacy ofGPP-targeting, as described in detail below.

The level of any one or more of the markers GLS, GOT2, GOT1, MDH1, ME1,Kras, GLUD1, NADP+, NADPH, GSSG, GSH, pyruvate, glutamine, glutamate,aspartate, αKG, NAD+, NADH, oxaloacetate, ROS (e.g., hydrogen peroxide,super oxide, hydroxyl radical, hypochlorous acid, nitric oxide, peroxylradical, and singlet oxygen), and malate may be determined in order todetermine the efficacy of GPP signaling. The skilled artisan willappreciate that other markers, e.g., subsequently, determined to beassociated with an enzyme-catalyzed reaction in the GPP are alsoencompassed by the present methods.

Typically, the level(s) of the marker(s) is (are) determined in a sampleobtained from a subject who is undergoing or has undergoneGPP-targeting. The level of the marker is compared to a control level.The control level of a marker typically is the level of the marker in asample obtained from the same subject prior to or at the beginning ofthe GPP-targeting. However, the control level can also be based on thelevel of the marker in a sample obtained from another subject orsubjects who is/are known to comprise an oncogenic Kras mutation, solong as that subject or those subjects is/are not undergoing or has/havenot undergone GPP-targeting, and preferably, although not necessarily,any other cancer therapies; such a control may be useful, e.g., if asubject's “prior to or at the beginning of the GPP-targeting” sample isnot available or was not obtained. In certain embodiments, such controlsubject(s) has (have) an oncogenic Kras-associated cancer.

Effective inhibition of the GPP will result in a change (i.e., increaseor decrease) in the level of the marker in the sample relative to thecontrol level. The control level may be predetermined, or may besimultaneously determined (e.g., measured at the same time the level inthe sample is measured).

In certain embodiments, the GPP-targeting is determined to have efficacyif the level of the marker is increased or decreased by a fold-change ofat least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least4.5 fold, at least 5, at least 5.5, at least 6, at least 6.5, at least7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, atleast 10, at least 15, at least 20, or more, compared to the marker'scontrol level. In a preferred embodiment, the level is increased ordecreased by at least 2-fold.

In certain embodiments, the control level can be the level in a sampleobtained from a healthy subject or subjects (i.e., subject or subjectswho have been determined to not comprise an oncogenic Kras mutation andpreferably who also do not have cancer). In those embodiments, theGPP-targeting is determined to have been effective if the level of themarker in the sample is the same, i.e., not significantly different(e.g., statistically and/or less than two-fold difference in level) thanthe control sample. In other words, if the level of the marker in thesubject is the same as the level in the control, then it is concludedthat the GPP-targeting was effective.

In a preferred embodiment, the marker or combination of markers isselected based on the specific target (e.g., enzyme(s) and/ormetabolite(s) of the therapy). As discussed in detail below, and asdemonstrated in the present Examples, inhibition of a particular enzymeor metabolite will most observably affect a specific set of markers thatare upstream and downstream of the particular target. The skilledartisan will know, based on the description of the GPP provided herein,including a detailed illustration of the pathway in FIG. 39, and thepresent Examples, how to determine whether the level of a particularmarker will increase or decrease when a given enzyme or metabolite istargeted by the therapy.

For clarity and illustrative purposes, the effect of inhibition ofcertain enzymes in the GPP on the expression levels of certainmetabolites in the pathway are exemplified in Table 2, below. However,it is to be understood that Table 2 provides non-limiting examples, andmay not show every metabolite that may be affected by inhibition of aparticular enzyme, and the skilled artisan will know, based on thedisclosure of the GPP (e.g., FIG. 18) and the Examples provided herein,what effect and/or how to determine what effect, if any, inhibition ofother enzymes and/or metabolites associated with an enzyme-catalyzedreaction in the GPP will have on the level of the above-describedmarkers.

TABLE 2 Markers Regulated by GPP-Targeting of Specific Enzymes EnzymeInhibited: KRAS GLS GOT2 GOT1 MDH1 ME1 Markers Gln Gln NAD+ Asp OAA AspWith OAA NAD+ NADP+ αKG Asp OAA Increased NAD+ NADP+ GSSG NAD+ NAD+Malate Expression NADP+ GSSG ROS NADP+ NADP+ NADP+ Level* GSSG ROS GSSGGSSG GSSG ROS ROS ROS ROS Markers αKG Asp Asp Malate Malate Gln With AspMalate NADH Glu NADH αKG Decreased Malate Glu Pyruvate OAA Pyruvate GluExpression Glu OAA NADPH NADH NADPH Pyruvate Level* NADH NADH GSHPyruvate GSH NADPH Pyruvate Pyruvate NADPH GSH NADPH NADPH GSH GSH GSH*compared to a control level of the marker (level prior to inhibition ofthe target enzyme)

In a preferred embodiment, the method for determining the efficacy ofGPP-targeting comprises determining the level of one or more markersselected from the group consisting of NADP+, NADPH, GSSG, GSH, pyruvate,and ROS in a sample obtained from a subject who is undergoing or hasundergone the GPP-targeting, and concluding that the GPP-targeting waseffective if the level of one or more of the markers NADP+, GSSG and ROSis increased, relative to each marker's control level, or if the levelof one or more of the markers NADPH, GSH and pyruvate is decreased,relative to each marker's control level; or concluding that theGPP-targeting was not effective if the level of one or more of themarkers NADP+, GSSG, and ROS is not increased, relative to each marker'scontrol level, or if the level of one or more of the markers NADPH, GSHand pyruvate is not decreased, relative to each marker's control level.The method can also comprise determining the level of 2 or more, 3 ormore, 4 or more, 5 or more, or all 6 or the above-described markers. Themethod can further comprise determining the level of at least oneadditional marker selected from the group consisting of glutamine,glutamate, aspartate, αKG, NAD+, NADH, oxaloacetate, malate, MDH1, andME1.

In certain embodiments, the ratio of NADP+/NADH and/or the ratio ofGSSG/GSH and/or the level of ROS is/are determined. In one embodiment,two or more of the ratio of NADP+/NADH, the ratio of GSSG/GSH, and thelevel of ROS are determined. In another embodiment, the ratio ofNADP+/NADH, and the ratio of GSSG/GSH and the level of ROS aredetermined.

The skilled artisan will readily appreciate that the levels of anycombination of the above-described markers can be determined accordingto the present methods.

In certain embodiments, methods for determining the efficacy ofGPP-targeting are provided, wherein the GPP-targeting comprisesinhibiting the enzyme GOT2. In those embodiments, it may be concludedthat the GPP-targeting was effective if the level of one or more of themarkers NADP+, GSSG and ROS is increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is decreased, relative to each marker's control level;and, optionally, if the level of at least one of the markers selectedfrom the group consisting of NAD+ and NADH is altered relative to eachmarker's control level. On the other hand, it may be concluded that theGPP-targeting was not effective if the level of one or more of themarkers NADP+, GSSG and ROS is not increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is not decreased, relative to each marker's controllevel; and, optionally, if the level of at least one of the markersselected from the group consisting of NAD+ NADH is not altered relativeto each marker's control level. The skilled artisan will readilyappreciate that the levels of any combination of the above-describedmarkers can be determined in order to determine the efficacy of GPPtargeting comprising inhibiting the enzyme GOT2.

In another embodiment, methods for determining the efficacy ofGPP-targeting are provided, wherein the GPP-targeting comprisesinhibiting the enzyme Kras. In that embodiment, it may be concluded thatthe GPP-targeting was effective if the level of one or more of themarkers NADP+, GSSG and ROS is increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is decreased, relative to each marker's control level;and/or if the level of at least one of the markers selected from thegroup consisting of glutamine, oxaloacetate, and NAD+ is increasedrelative to its control level; and/or if the level of at least one ofthe markers selected from the group consisting of aspartate, αKG,malate, MDH1, and ME1 is decreased relative to each marker's controllevel. On the other hand, it may be concluded that the GPP-targeting wasnot effective if the level of one or more of the markers NADP+, GSSG andROS is not increased, relative to each marker's control level, and/or ifthe level of one or more of the markers NADPH, GSH, and pyruvate is notdecreased, relative to each marker's control level; and, optionally, ifthe level of at least one of the markers selected from the groupconsisting of glutamine, oxaloacetate, and NAD+ is not increasedrelative to its control level; and/or if the level of at least one ofthe markers selected from the group consisting of aspartate, αKG,malate, MDH1, and ME1 is not decreased relative to each marker's controllevel.

In still another embodiment, a method for determining the efficacy ofoncogenic Kras inhibition in a subject is provided. While not intendingto be bound by any one particular theory or mechanism of action, themethod is based, at least in part, on the present discovery thatinhibition of Kras leads to decreased expression of the enzymes MDH1 andME1. Thus, in certain embodiments, the method comprises determining theexpression level of at least one of the enzymes MDH1 and ME1 in a sampleobtained from a subject who is undergoing or has undergone the oncogenicKras inhibition; and concluding that the oncogenic Kras inhibition waseffective if the expression level of the at least one enzyme isdecreased compared to a control level of the enzyme; or concluding thatthe oncogenic Kras inhibition was not effective if the expression levelof the at least one enzyme is not decreased compared to a control levelof the enzyme.

In other embodiments, methods for determining the efficacy ofGPP-targeting are provided, wherein the GPP-targeting comprisesinhibiting the enzyme GOT1. In those embodiments, it may be concludedthat the GPP-targeting was effective if the level of one or more of themarkers NADP+, GSSG and ROS is increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is decreased, relative to each marker's control level;and, optionally, if the level of at least one of the markers selectedfrom the group consisting of aspartate, αKG, and NAD+ is increasedrelative to each marker's control level; and/or if the level of at leastone of the markers selected from the group consisting of oxaloacetate,malate, glutamate, and NADH is decreased relative to each marker'scontrol level. On the other hand, it may be concluded that theGPP-targeting was not effective if the level of one or more of themarkers NADP+, GSSG and ROS is not increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is not decreased, relative to each marker's controllevel; and, optionally, if the level of at least one of the markersselected from the group consisting of aspartate, cKG, and NAD+ is notincreased relative to each marker's control level; and/or if the levelof at least one of the markers selected from the group consisting ofoxaloacetate, malate, glutamate, and NADH is not decreased relative toeach marker's control level.

In other embodiments, methods for determining the efficacy ofGPP-targeting are provided, wherein the GPP-targeting comprisesinhibiting the enzyme MDH1. In those embodiments, it may be concludedthat the GPP-targeting was effective if the level of one or more of themarkers NADP+, GSSG and ROS is increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is decreased, relative to each marker's control level;and, optionally, if the level of at least one of the markers selectedfrom the group consisting of oxaloacetate and NAD+ is increased relativeto each marker's control level; and/or if the level of at least one ofthe markers selected from the group consisting of malate and NADH isdecreased relative to each marker's control level. On the other hand, itmay be concluded that the GPP-targeting was not effective if the levelof one or more of the markers NADP+, GSSG and ROS is not increased,relative to each marker's control level, and/or if the level of one ormore of the markers NADPH, GSH, and pyruvate is not decreased, relativeto each marker's control level; and, optionally, if the level of atleast one of the markers selected from the group consisting ofoxaloacetate and NAD+ is not increased relative to each marker's controllevel; and/or if the level of at least one of the markers selected fromthe group consisting of malate and NADH is not decreased relative toeach marker's control level.

In other embodiments, methods for determining the efficacy ofGPP-targeting are provided, wherein the GPP-targeting comprisesinhibiting the enzyme GLS. In those embodiments, it may be concludedthat the GPP-targeting was effective if the level of one or more of themarkers NADP+, GSSG and ROS is increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is decreased, relative to each marker's control level;and, optionally, if the level of at least one of the markers selectedfrom the group consisting of glutamine and NAD+ is increased relative toeach marker's control level, or if the level of at least one of themarkers selected from the group consisting of aspartate, oxaloacetate,malate, glutamate, and NADH is decreased relative to each marker'scontrol level. On the other hand, it may be concluded that theGPP-targeting was not effective if the level of one or more of themarkers NADP+, GSSG and ROS is not increased, relative to each marker'scontrol level, and/or if the level of one or more of the markers NADPH,GSH, and pyruvate is not decreased, relative to each marker's controllevel; and, optionally, if the level of at least one of the markersselected from the group consisting of glutamine and NAD+ is notincreased relative to each marker's control level, and/or if the levelof at least one of the markers selected from the group consisting ofaspartate, oxaloacetate, malate, glutamate, and NADH is not decreasedrelative to each marker's control level.

In yet another embodiment, a method for determining the efficacy ofGPP-targeting in a subject comprising an oncogenic Kras mutation (e.g.,a subject with a cancer associated with an oncogenic Kras mutation) isprovided, wherein the GPP-targeting comprises targeting one or more ofthe enzymes catalyzing the conversion of glutamine to aspartate (e.g.GLS, GOT2). While not intending to be bound by any one particular theoryor mechanism of action, the present method is based, at least in part,on the present discovery that glutamate is converted to aspartate in theGPP, rather than to αKG via the canonical pathway mediated by GLUD1.Thus, effective inhibition of the enzymes catalyzing the conversion ofglutamine to aspartate can be determined by testing for alterations ofany of the metabolites downstream of and including aspartate in the GPP.Preferably, the method comprises determining the level of at least onemarker selected from the group consisting of GSSG, GSH, aspartate, αKG,NADP+, NADPH, NAD+, NADH, pyruvate, oxaloacetate, and malate in a sampleobtained from a subject who is undergoing or has undergone theGPP-targeting. Further, it may be concluded that the GPP-targeting waseffective if the level of one or more of GSSG, GSH, aspartate, αKG,NADP+, NADPH, NAD+, NADH, pyruvate, oxaloacetate, and malate is alteredrelative to each marker's control level.

In another method, a method for determining the efficacy of oncogenicKras inhibition in a subject having a cell having an oncogenic Krasmutation is provided, wherein the method includes determining theexpression level of at least one of the enzymes GLUD1 and GOT1 in asample obtained from a subject who is undergoing or has undergone theoncogenic Kras inhibition; and concluding that the oncogenic Krasinhibition was effective if the expression level of GOT1 is decreasedcompared to a control level of the enzyme and/or if the expression levelof GLUD1 is increased compared to a control level of the enzyme; orconcluding that the oncogenic Kras inhibition was not effective if theexpression level of GOT1 is not decreased compared to a control level ofthe enzyme and/or if the expression level of GLUD1 is not increasedcompared to a control level of the enzyme. In certain aspects of themethod, the ratio of GLUD1 and GOT1 can be determined.

In certain methods disclosed herein, the method includes concluding thatthe GPP-targeting (e.g., with an inhibitor of GOT1 or MDH1) was noteffective if the level of aspartate is not increased relative to acontrol level; or concluding that the GPP-targeting was effective if thelevel of aspartate is increased relative to a control level.

Also provided herein is a method for determining the efficacy ofglutamine to pyruvate pathway (GPP)-targeting in a subject having anoncogenic Kras mutation, and the GPP-targeting comprises inhibitingGOT1, MDH1, or ME1, the method including: determining the level ofaspartate in a sample obtained from a subject who is undergoing or hasundergone the GPP-targeting; and concluding that the GPP-targeting waseffective if the level of aspartate is increased relative to a controllevel; or concluding that the GPP-targeting was not effective if thelevel of aspartate is not increased relative to a control level.

In any of the above-disclosed methods for determining the efficacy ofGPP-targeting in a subject having an oncogenic Kras mutation (e.g., asubject with a cancer associated with an oncogenic Kras mutation), themethod can include further steps. For example, in one embodiment, themethod can further include continuing the GPP-targeting of the subject,at the same dose or frequency or at a lower dose or frequency, if, forexample, it is concluded that the GPP-targeting prior to the efficacydetermination was at least partially effective. Moreover, the method canfurther include decreasing the dose and/or frequency or altogetherdiscontinuing GPP-targeting in the subject, if, for example, it isconcluded that the targeting was curatively effective. However, if, forexample, it is concluded that GPP-targeting was not effective, it can bediscontinued. Alternatively, if it was not effective, or was notoptimal, the method can further include the step of administering adifferent treatment or repeating the GPP-targeting in the subject and/oradjusting the dose and/or frequency of GPP targeting. Differenttreatments are known in art and include those described herein, such assurgery, chemotherapy, and radiotherapy. In other embodiments, if, forexample, it is concluded that GPP-targeting was not effective, or wasnot optimal, the method can further include the step of administering acombination treatment to the subject, e.g., a treatment targeting atleast two members (e.g., metabolite or enzyme) of the GPP or anyGPP-targeting in combination with one or more of the above-mentioned“different treatments.”

VIII. Design of Metabolite and Enzyme Inhibitors

Design of inhibitors of the metabolites disclosed herein, such as, e.g.,those described above, will depend on the specific metabolite beingtargeted. Non-limiting examples of such inhibitors include, e.g., smallmolecules and non-functional binding proteins, which can trap themetabolite and prevent its function. Hydrogen peroxide, for example, andwithout limitation, can be used to antagonize GSH, NADH, and NADPH.Further, aminooxyacentate (AOA), as disclosed in the present Examples,and in Wise, D. R. et al. (2008) Proc Natl Acad Sci USA 105,18782-18787], inhibits transaminases (e.g., GOT1 and GOT2). The skilledartisan will understand how to design such inhibitors, based on methodswell known in the art.

Methods for designing inhibitors of the enzymes of the invention (e.g.,GLS, GOT2, GOT1, MDH1, ME1, etc.) are well known in the art. Thefollowing are thus provided as non-limiting examples of such inhibitors;the skilled artisan will understand that other inhibitors that decreasethe level (e.g., expression or activity) of a target of the inventionare also encompassed by the present methods.

i. Antisense Nucleic Acids

Antisense oligonucleotides can be used to inhibit the expression of atarget polypeptide of the invention (e.g., Gfpt1, RPIA, RPE, etc.).Antisense oligonucleotides typically comprise from about 5 nucleotidesto about 30 nucleotides in length, preferably from about 10 to about 25nucleotides in length, and more preferably from about 20 to about 25nucleotides in length. For a general discussion of antisense technology,see, e.g., Antisense DNA and RNA, (Cold Spring Harbor Laboratory, D.Melton, ed., 1988).

Appropriate chemical modifications of the inhibitors are made to ensurestability of the antisense oligonucleotide, as described below. Changesin the nucleotide sequence and/or in the length of the antisenseoligonucleotide can be made to ensure maximum efficiency andthermodynamic stability of the inhibitor. Such sequence and/or lengthmodifications are readily determined by one of ordinary skill in theart.

The antisense oligonucleotides can be DNA or RNA or chimeric mixtures,or derivatives or modified versions thereof, and can be single-strandedor double-stranded. Thus, for example, in the antisense oligonucleotidesset forth in herein, when a sequence includes thymidine residues, one ormore of the thymidine residues may be replaced by uracil residues and,conversely, when a sequence includes uracil residues, one or more of theuracil residues may be replaced by thymidine residues.

Antisense oligonucleotides comprise sequences complementary to at leasta portion of the corresponding target polypeptide. However, 100%sequence complementarity is not required so long as formation of astable duplex (for single stranded antisense oligonucleotides) ortriplex (for double stranded antisense oligonucleotides) can beachieved. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense oligonucleotides.Generally, the longer the antisense oligonucleotide, the more basemismatches with the corresponding nucleic acid target can be tolerated.One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Antisense nucleic acid molecules can be encoded by a recombinant genefor expression in a cell (see, e.g., U.S. Pat. Nos. 5,814,500 and5,811,234), or alternatively they can be prepared synthetically (see,e.g., U.S. Pat. No. 5,780,607).

The antisense oligonucleotides can be modified at the base moiety, sugarmoiety, or phosphate backbone, or a combination thereof. In oneembodiment, the antisense oligonucleotide comprises at least onemodified sugar moiety, e.g., a sugar moiety selected from arabinose,2-fluoroarabinose, xylulose, and hexose.

In another embodiment, the antisense oligonucleotide comprises at leastone modified phosphate backbone selected from a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof. Examples include, without limitation,phosphorothioate antisense oligonucleotides (e.g., an antisenseoligonucleotide phosphothioate modified at 3′ and 5′ ends to increaseits stability) and chimeras between methylphosphonate and phosphodiesteroligonucleotides. These oligonucleotides provide good in vivo activitydue to solubility, nuclease resistance, good cellular uptake, ability toactivate RNase H, and high sequence selectivity.

Other examples of synthetic antisense oligonucleotides includeoligonucleotides that contain phosphorothioates, phosphotriesters,methyl phosphonates, short chain alkyl, or cycloalkyl intersugarlinkages or short chain heteroatomic or heterocyclic intersugarlinkages. Most preferred are those with CH2-NH—O—CH2, CH2-N(CH3)-O—CH2,CH2-O—N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones(where phosphodiester is O—PO2-O—CH2). U.S. Pat. No. 5,677,437 describesheteroaromatic oligonucleoside linkages. Nitrogen linkers or groupscontaining nitrogen can also be used to prepare oligonucleotide mimics(U.S. Pat. Nos. 5,792,844 and 5,783,682). U.S. Pat. No. 5,637,684describes phosphoramidate and phosphorothioamidate oligomeric compounds.

In other embodiments, such as the peptide-nucleic acid (PNA) backbone,the phosphodiester backbone of the oligonucleotide may be replaced witha polyamide backbone, the bases being bound directly or indirectly tothe aza nitrogen atoms of the polyamide backbone (Nielsen et al.,Science 1991;254:1497). Other synthetic oligonucleotides may containsubstituted sugar moieties comprising one of the following at the 2′position: OH, SH, SCH3, F, OCN, O(CH2)nNH2 or O(CH2)nCH3 where n is from1 to about 10; C1 to C10 lower alkyl, substituted lower alkyl, alkarylor aralkyl; Cl; Br; CN; CF3; OCF3; O-; S-, or N-alkyl; O-, S-, orN-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl;a fluorescein moiety; an RNA cleaving group; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.

Oligonucleotides may also have sugar mimetics such as cyclobutyls orother carbocyclics in place of the pentofuranosyl group. Nucleotideunits having nucleosides other than adenosine, cytidine, guanosine,thymidine and uridine may be used, such as inosine. In otherembodiments, locked nucleic acids (LNA) can be used (reviewed in, e.g.,Jepsen and Wengel, Curr. Opin. Drug Discov. Devel. 2004; 7:188-194;Crinelli et al., Curr. Drug Targets 2004; 5:745-752). LNA are nucleicacid analog(s) with a 2′-O, 4′-C methylene bridge. This bridge restrictsthe flexibility of the ribofuranose ring and locks the structure into arigid C3-endo conformation, conferring enhanced hybridizationperformance and exceptional biostability. LNA allows the use of veryshort oligonucleotides (less than 10 bp) for efficient hybridization invivo.

In one embodiment, an antisense oligonucleotide can comprise at leastone modified base moiety selected from a group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

In another embodiment, the antisense oligonucleotide can includeα-anomeric oligonucleotides. An α-anomeric oligonucleotide formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 1987; 15:6625-6641).

Oligonucleotides may have morpholino backbone structures (U.S. Pat. No.5,034,506). Thus, in yet another embodiment, the antisenseoligonucleotide can be a morpholino antisense oligonucleotide (i.e., anoligonucleotide in which the bases are linked to 6-membered morpholinerings, which are connected to other morpholine-linked bases vianon-ionic phosphorodiamidate intersubunit linkages). Morpholinooligonucleotides are highly resistant to nucleases and have goodtargeting predictability, high in-cell efficacy and high sequencespecificity (U.S. Pat. No. 5,034,506; Summerton, Biochim. Biophys. Acta1999; 1489:141-158; Summerton and Weller, Antisense Nucleic Acid DrugDev. 1997; 7:187-195; Arora et al., J. Pharmacol. Exp. Ther.2000;292:921-928; Qin et al., Antisense Nucleic Acid Drug Dev. 2000;10:11-16; Heasman et al., Dev. Biol. 2000; 222:124-134; Nasevicius andEkker, Nat. Genet. 2000; 26:216-220).

Antisense oligonucleotides may be chemically synthesized, for exampleusing appropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Antisense nucleic acidoligonucleotides can also be produced intracellularly by transcriptionfrom an exogenous sequence. For example, a vector can be introduced invivo such that it is taken up by a cell within which the vector or aportion thereof is transcribed to produce an antisense RNA. Such avector can remain episomal or become chromosomally integrated, so longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in mammalian cells. Inanother embodiment, “naked” antisense nucleic acids can be delivered toadherent cells via “scrape delivery”, whereby the antisenseoligonucleotide is added to a culture of adherent cells in a culturevessel, the cells are scraped from the walls of the culture vessel, andthe scraped cells are transferred to another plate where they areallowed to re-adhere. Scraping the cells from the culture vessel wallsserves to pull adhesion plaques from the cell membrane, generating smallholes that allow the antisense oligonucleotides to enter the cytosol.

ii. RNAi

Reversible short inhibition of a target polypeptide (e.g., Gfpt1, RPIA,RPE, etc.) of the invention may also be useful. Such inhibition can beachieved by use of siRNAs. RNA interference (RNAi) technology preventsthe expression of genes by using small RNA molecules such as smallinterfering RNAs (siRNAs). This technology in turn takes advantage ofthe fact that RNAi is a natural biological mechanism for silencing genesin most cells of many living organisms, from plants to insects tomammals (McManus et al., Nature Reviews Genetics, 2002, 3(10) p. 737).RNAi prevents a gene from producing a functional protein by ensuringthat the molecule intermediate, the messenger RNA copy of the gene isdestroyed siRNAs can be used in a naked form and incorporated in avector, as described below.

RNA interference (RNAi) is a process of sequence-specificpost-transcriptional gene silencing by which double stranded RNA (dsRNA)homologous to a target locus can specifically inactivate gene functionin plants, fungi, invertebrates, and vertebrates, including mammals(Hammond et al., Nature Genet. 2001;2:110-119; Sharp, Genes Dev.1999;13:139-141). This dsRNA-induced gene silencing is mediated by shortdouble-stranded small interfering RNAs (siRNAs) generated from longerdsRNAs by ribonuclease III cleavage (Bernstein et al., Nature 2001;409:363-366 and Elbashir et al., Genes Dev. 2001; 15:188-200).RNAi-mediated gene silencing is thought to occur via sequence-specificRNA degradation, where sequence specificity is determined by theinteraction of an siRNA with its complementary sequence within a targetRNA (see, e.g., Tuschl, Chem. Biochem. 2001; 2:239-245).

For mammalian systems, RNAi commonly involves the use of dsRNAs that aregreater than 500 bp; however, it can also be activated by introductionof either siRNAs (Elbashir, et al., Nature 2001; 411: 494-498) or shorthairpin RNAs (shRNAs) bearing a fold back stem-loop structure (Paddisonet al., Genes Dev. 2002; 16: 948-958; Sui et al., Proc. Natl. Acad. Sci.USA 2002; 99:5515-5520; Brummelkamp et al., Science 2002; 296:550-553;Paul et al., Nature Biotechnol. 2002; 20:505-508).

The siRNAs are preferably short double stranded nucleic acid duplexescomprising annealed complementary single stranded nucleic acidmolecules. Preferably, the siRNAs are short dsRNAs comprising annealedcomplementary single strand RNAs. siRNAs may also comprise an annealedRNA:DNA duplex, wherein the sense strand of the duplex is a DNA moleculeand the antisense strand of the duplex is a RNA molecule.

Preferably, each single stranded nucleic acid molecule of the siRNAduplex is of from about 19 nucleotides to about 27 nucleotides inlength. In preferred embodiments, duplexed siRNAs have a 2 or 3nucleotide 3′ overhang on each strand of the duplex. In preferredembodiments, siRNAs have 5′-phosphate and 3′-hydroxyl groups.

RNAi molecules may include one or more modifications, either to thephosphate-sugar backbone or to the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one heteroatom other than oxygen, such as nitrogen or sulfur. Inthis case, for example, the phosphodiester linkage may be replaced by aphosphothioester linkage. Similarly, bases may be modified to block theactivity of adenosine deaminase. Where the RNAi molecule is producedsynthetically, or by in vitro transcription, a modified ribonucleosidemay be introduced during synthesis or transcription. The skilled artisanwill understand that many of the modifications described above forantisense oligonucleotides may also be made to RNAi molecules. Suchmodifications are well known in the art.

siRNAs may be introduced to a target cell as an annealed duplex siRNA,or as single stranded sense and antisense nucleic acid sequences that,once within the target cell, anneal to form the siRNA duplex.Alternatively, the sense and antisense strands of the siRNA may beencoded on an expression construct that is introduced to the targetcell. Upon expression within the target cell, the transcribed sense andantisense strands may anneal to reconstitute the siRNA.

shRNAs typically comprise a single stranded “loop” region connectingcomplementary inverted repeat sequences that anneal to form a doublestranded “stem” region. Structural considerations for shRNA design arediscussed, for example, in McManus et al., RNA 2002; 8:842-850. Incertain embodiments the shRNA may be a portion of a larger RNA molecule,e.g., as part of a larger RNA that also contains U6 RNA sequences (Paulet al., supra).

In preferred embodiments, the loop of the shRNA is from about 1 to about9 nucleotides in length. In preferred embodiments the double strandedstem of the shRNA is from about 19 to about 33 base pairs in length. Inpreferred embodiments, the 3′ end of the shRNA stem has a 3′ overhang.In particularly preferred embodiments, the 3′ overhang of the shRNA stemis from 1 to about 4 nucleotides in length. In preferred embodiments,shRNAs have 5′-phosphate and 3′-hydroxyl groups.

Although RNAi molecules preferably contain nucleotide sequences that arefully complementary to a portion of the target nucleic acid, 100%sequence complementarity between the RNAi probe and the target nucleicacid is not required.

Similar to the above-described antisense oligonucleotides, RNAimolecules can be synthesized by standard methods known in the art, e.g.,by use of an automated synthesizer. RNAs produced by such methodologiestend to be highly pure and to anneal efficiently to form siRNA duplexesor shRNA hairpin stem-loop structures. Following chemical synthesis,single stranded RNA molecules are deprotected, annealed to form siRNAsor shRNAs, and purified (e.g., by gel electrophoresis or HPLC).Alternatively, standard procedures may be used for in vitrotranscription of RNA from DNA templates carrying RNA polymerase promotersequences (e.g., T7 or SP6 RNA polymerase promoter sequences). Efficientin vitro protocols for preparation of siRNAs using T7 RNA polymerasehave been described (Donze and Picard, Nucleic Acids Res. 2002; 30:e46;and Yu et al., Proc. Natl. Acad. Sci. USA 2002; 99:6047-6052).Similarly, an efficient in vitro protocol for preparation of shRNAsusing T7 RNA polymerase has been described (Yu et al., supra). The senseand antisense transcripts may be synthesized in two independentreactions and annealed later, or may be synthesized simultaneously in asingle reaction.

RNAi molecules may be formed within a cell by transcription of RNA froman expression construct introduced into the cell. For example, both aprotocol and an expression construct for in vivo expression of siRNAsare described in Yu et al., supra. The delivery of siRNA to tumors canpotentially be achieved via any of several gene delivery “vehicles” thatare currently available. These include viral vectors, such asadenovirus, lentivirus, herpes simplex virus, vaccinia virus, andretrovirus, as well as chemical-mediated gene delivery systems (forexample, liposomes), or mechanical DNA delivery systems (DNA guns). Theoligonucleotides to be expressed for such siRNA-mediated inhibition ofgene expression would be between 18 and 28 nucleotides in length.Protocols and expression constructs for in vivo expression of shRNAshave been described (Brummelkamp et al., Science 2002; 296:550-553; Suiet al., supra; Yu et al., supra; McManus et al., supra; Paul et al.,supra).

The expression constructs for in vivo production of RNAi moleculescomprise RNAi encoding sequences operably linked to elements necessaryfor the proper transcription of the RNAi encoding sequence(s), includingpromoter elements and transcription termination signals. Preferredpromoters for use in such expression constructs include thepolymerase-III HI-RNA promoter (see, e.g., Brummelkamp et al., supra)and the U6 polymerase-III promoter (see, e.g., Sui et al., supra; Paul,et al. supra; and Yu et al., supra). The RNAi expression constructs canfurther comprise vector sequences that facilitate the cloning of theexpression constructs. Standard vectors are known in the art (e.g.,pSilencer 2.0-U6 vector, Ambion Inc., Austin, Tex.).

iii. Ribozyme Inhibition

The level of expression of a target polypeptide of the invention canalso be inhibited by ribozymes designed based on the nucleotide sequencethereof.

Ribozymes are enzymatic RNA molecules capable of catalyzing thesequence-specific cleavage of RNA (for a review, see Rossi, CurrentBiology 1994;4:469-471). The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage event.The composition of ribozyme molecules must include: (i) one or moresequences complementary to the target RNA; and (ii) a catalytic sequenceresponsible for RNA cleavage (see, e.g., U.S. Pat. No. 5,093,246).

The use of hammerhead ribozymes is preferred. Hammerhead ribozymescleave RNAs at locations dictated by flanking regions that formcomplementary base pairs with the target RNA. The sole requirement isthat the target RNA has the following sequence of two bases: 5′-UG-3′.The construction of hammerhead ribozymes is known in the art, anddescribed more fully in Myers, Molecular Biology and Biotechnology: AComprehensive Desk Reference, VCH Publishers, New York, 1995 (seeespecially FIG. 4, page 833) and in Haseloff and Gerlach, Nature 1988;334:585-591.

As in the case of antisense oligonucleotides, ribozymes can be composedof modified oligonucleotides (e.g., for improved stability, targeting,etc.). These can be delivered to cells which express the targetpolypeptide in vivo. A preferred method of delivery involves using a DNAconstruct “encoding” the ribozyme under the control of a strongconstitutive pol III or pol II promoter, so that transfected cells willproduce sufficient quantities of the ribozyme to catalyze cleavage ofthe target mRNA encoding the target polypeptide. However, becauseribozymes, unlike antisense molecules, are catalytic, a lowerintracellular concentration may be required to achieve an adequate levelof efficacy.

Ribozymes can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules, as discussed above. Ribozymetechnology is described further in Intracellular Ribozyme Applications:Principals and Protocols, Rossi and Couture eds., Horizon ScientificPress, 1999.

iv. Triple Helix Forming Oligonucleotides (TFOs)

Nucleic acid molecules useful to inhibit expression level of a targetpolypeptide of the invention via triple helix formation are preferablycomposed of deoxynucleotides. The base composition of theseoligonucleotides is typically designed to promote triple helix formationvia Hoogsteen base pairing rules, which generally require sizeablestretches of either purines or pyrimidines to be present on one strandof a duplex. Nucleotide sequences may be pyrimidine-based, resulting inTAT and CGC triplets across the three associated strands of theresulting triple helix. The pyrimidine-rich molecules provide basecomplementarity to a purine-rich region of a single strand of the duplexin a parallel orientation to that strand. In addition, nucleic acidmolecules may be chosen that are purine-rich, e.g., those containing astretch of G residues. These molecules will form a triple helix with aDNA duplex that is rich in GC pairs, in which the majority of the purineresidues are located on a single strand of the targeted duplex,resulting in GGC triplets across the three strands in the triplex.

Alternatively, sequences can be targeted for triple helix formation bycreating a so-called “switchback” nucleic acid molecule. Switchbackmolecules are synthesized in an alternating 5′-3′, 3′-5′ manner, suchthat they base pair with first one strand of a duplex and then theother, eliminating the necessity for a sizeable stretch of eitherpurines or pyrimidines to be present on one strand of a duplex.

Similarly to RNAi molecules, antisense oligonucleotides, and ribozymes,described above, triple helix molecules can be prepared by any methodknown in the art. These include techniques for chemically synthesizingoligodeoxyribonucleotides and oligoribonucleotides such as, e.g., solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro or in vivo transcription of DNA sequences“encoding” the particular RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.See, Nielsen, P. E. “Triple Helix: Designing a New Molecule of Life”,Scientific American, December, 2008; Egholm, M., et al. “PNA Hybridizesto Complementary Oligonucleotides Obeying the Watson-Crick HydrogenBonding Rules.” (1993) Nature, 365, 566-568; Nielsen, P. E. ‘PNATechnology’. Mol Biotechnol. 2004;26:233-48.

v. Antibodies and Aptamers

The enzymes described herein, e.g., GOT1, GOT2, MDH1, ME1, can beinhibited (e.g. the level can be reduced) by the administration to orexpression in a subject or a cell or tissue thereof, of blockingantibodies or aptamers against the enzyme.

Antibodies, or their equivalents and derivatives, e.g., intrabodies, orother antagonists of the enzyme, may be used in accordance with thepresent methods for GPP-targeting. Methods for engineering intrabodies(intracellular single chain antibodies) are well known. Intrabodies arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13; Lo et al.(2009) Handb Exp Pharmacol. 181:343-73; Marasco, W. A. (1997) GeneTherapy 4:11-15; see also, U.S. Pat. Appln. Pub. No. 2001/0024831 by DerMaur et al. and U.S. Pat. No. 6,004,940 by Marasco et al.).

Administration of a suitable dose of the antibody or the antagonist(e.g., aptamer) may serve to block the level (expression or activity) ofthe enzyme in order to block the GPP, and/or e.g., inhibit growth of acell, e.g., cancer cell, comprising an oncogenic Kras mutation.

In addition to using antibodies to inhibit the levels and/or activity ofthe enzyme, it may also be possible to use other forms of inhibitors.For example, it may be possible to identify antagonists thatfunctionally inhibit the target enzyme (e.g., GOT1, GOT2, ME1, MDH1,etc.). In addition, it may also be possible to interfere with theinteraction of the enzyme with its substrate. Other suitable inhibitorswill be apparent to the skilled person.

The antibody (or other inhibitors or intrabody) can be administered by anumber of methods. One method is set forth by Marasco and Haseltine inPCT WO 94/02610. This method discloses the intracellular delivery of agene encoding the intrabody. In one embodiment, a gene encoding a singlechain antibody is used. In another embodiment, the antibody wouldcontain a nuclear localization sequence. By this method, one canintracellularly express an antibody, which can block activity of theenzyme in desired cells.

Aptamers are oligonucleic acid or peptide molecules that bind to aspecific target molecule. Aptamers can be used to inhibit geneexpression and to interfere with protein interactions and activity.Nucleic acid aptamers are nucleic acid species that have been engineeredthrough repeated rounds of in vitro selection (e.g. by SELEX (systematicevolution of ligands by exponential enrichment)) to bind to variousmolecular targets such as small molecules, proteins, nucleic acids, andeven cells, tissues and organisms. Peptide aptamers consist of avariable peptide loop attached at both ends to a protamersein scaffold.Aptamers are useful in biotechnological and therapeutic applications asthey offer molecular recognition properties that rival that ofantibodies. Aptamers can be engineered completely in a test tube, arereadily produced by chemical synthesis, possess desirable storageproperties, and elicit little or no immunogenicity in therapeuticapplication. Aptamers can be produced using the methodology disclosed ina U.S. Pat. No. 5,270,163 and WO 91/19813.

vi. Small Molecules

Chemical agents, referred to in the art as “small molecule” compoundsare typically organic, non-peptide molecules, having a molecular weightless than 10,000 Da, preferably less than 5,000 Da, more preferably lessthan 1,000 Da, and most preferably less than 500 Da. This class ofmodulators includes chemically synthesized molecules, for instance,compounds from combinatorial chemical libraries. Synthetic compounds maybe rationally designed or identified utilizing the screening methodsdescribed below. Methods for generating and obtaining small moleculesare well known in the art (Schreiber, Science 2000; 151:1964-1969;Radmann et al., Science 2000; 151:1947-1948).

IX. Administration

Compositions and formulations comprising an inhibitor (or agonist) ofthe invention (e.g., an inhibitor or agonist of an enzyme or metaboliteassociated with an enzyme-catalyzed reaction in the GPP, can beadministered topically, parenterally, orally, by inhalation, as asuppository, or by other methods known in the art. The term “parenteral”includes injection (for example, intravenous, intraperitoneal, epidural,intrathecal, intramuscular, intraluminal, intratracheal orsubcutaneous). Preferred routes of administration are oral andintravenous (IV), although intratumoral administration is also possible.

While it is possible to use an inhibitor or agonist or other compositionof the invention for therapy as is, it may be preferable to administer acomposition as a pharmaceutical formulation, e.g., in admixture with asuitable pharmaceutical excipient, diluent, or carrier selected withregard to the intended route of administration and standardpharmaceutical practice. Pharmaceutical formulations comprise at leastone active compound, or a pharmaceutically acceptable derivativethereof, in association with a pharmaceutically acceptable excipient,diluent, and/or carrier. The excipient, diluent and/or carrier must be“acceptable,” as defined above.

Administration of a composition of the invention (e.g., inhibitor oragonist) can be once a day, twice a day, or more often. Frequency may bedecreased during a treatment maintenance phase of the disease ordisorder, e.g., once every second or third day instead of every day ortwice a day. The dose and the administration frequency will depend onthe clinical signs, which confirm maintenance of the remission phase,with the reduction or absence of at least one or more preferably morethan one clinical signs of the acute phase known to the person skilledin the art. More generally, dose and frequency will depend in part onrecession of pathological signs and clinical and subclinical symptoms ofa disease condition or disorder contemplated for treatment with thepresent compounds.

It will be appreciated that the amount of a composition of the invention(e.g. inhibitor or agonist) required for use in treatment will vary withthe route of administration, the nature of the condition for whichtreatment is required, and the age, body weight and condition of thepatient, and will be ultimately at the discretion of the attendantphysician or veterinarian. Compositions will typically contain aneffective amount of the active agent(s), alone or in combination.Preliminary doses can be determined according to animal tests, and thescaling of dosages for human administration can be performed accordingto art-accepted practices.

Length of treatment, i.e., number of days, will be readily determined bya physician treating the subject; however the number of days oftreatment may range from 1 day to about 20 days. As provided by thepresent methods, the efficacy of treatment can be monitored during thecourse of treatment to determine whether the treatment has beensuccessful, or whether additional (or modified) treatment is necessary.

X. Kits

In certain embodiments, kits are provided for use in GPP-targeting,e.g., for the treatment of a cancer associated with an oncogenic Krasmutation. For example, a kit comprising an inhibitor of ME1 and one ormore inhibitors of one or more of the enzymes selected from the groupconsisting of Kras, GOT2, GLS, GOT1, and MDH1 is provided. In anotherembodiment, a kit comprising an inhibitor of GLS and one or moreinhibitors of one or more of the enzymes selected from the groupconsisting of Kras, GOT2, ME1, GOT1, and MDH1 is provided. In yetanother embodiment, a kit comprising an inhibitor of at least one of theenzymes selected from the group consisting of Kras, ME1, GLS, GOT1,GOT2, and MDH1, and an inhibitor of one or more metabolites associatedwith an enzyme-catalyzed reaction in the GPP is provided. The abovedescribed kits may comprise an inhibitor of two or more metabolitesassociated with an enzyme-catalyzed reaction in the GPP. In certainembodiments, the inhibitor(s) in the above-described kits can target ametabolite selected from the group consisting of glutamine, glutamate,aspartate, oxaloacetate, malate, pyruvate, NADH, NADPH, and GSH.

The kits, regardless of type, will generally comprise one or morecontainers into which the biological agents (e.g. inhibitors) are placedand, preferably, suitably aliquotted. The components of the kits may bepackaged either in aqueous media or in lyophilized form.

The above-described kits may come with instructions for using the kitfor GPP targeting, e.g., for treating or preventing a cancer associatedwith an oncogenic Kras mutation.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, recombinant DNA,immunology, cell biology and other related techniques within the skillof the art. See, e.g., Sambrook et al., (2001) Molecular Cloning: ALaboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.; Sambrook et al., (1989) Molecular Cloning: ALaboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.; Ausubel et al., eds. (2005) Current Protocols inMolecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacinoet al., eds. (2005) Current Protocols in Cell Biology. John Wiley andSons, Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocolsin Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al.,eds. (2005) Current Protocols in Microbiology, John Wiley and Sons,Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocols inProtein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; Enna et al.,eds. (2005) Current Protocols in Pharmacology John Wiley and Sons, Inc.:Hoboken, N.J.; Hames et al., eds. (1999) Protein Expression: A PracticalApproach. Oxford University Press: Oxford; Freshney (2000) Culture ofAnimal Cells: A Manual of Basic Technique. 4th ed. Wiley-Liss; amongothers. The Current Protocols listed above are updated several timesevery year.

The following examples are meant to illustrate, not limit, theinvention.

EXAMPLES Example 1 Materials and Methods

The following are the materials and methods used in the Examples setforth below.

Proliferation and clonogenic assays were performed as previouslydescribed (Yang, S. et al. Genes Dev 25, 717-729 (2011)). Tocharacterize Gln metabolism, targeted liquid chromatography-tandem massspectrometry was performed (Ying et al. supra). Briefly, cells weregrown in complete media and transferred into Gln-free media supplementedwith [U—¹³C₅]-Gln overnight (steady state) or for the indicatedtimepoints (flux analyses). For subcutaneous xenografts, PDAC cellsinfected with lentiviral shRNAs to suppress target gene expression weresuspended in 100 μl HBSS and injected subcutaneously into the lowerflank of NCr nude mice. For mouse xenografts, murine PDAC cells stablyinfected with a doxycycline-inducible GOT1 shRNA construct wereinjected. Animals were fed with doxycycline water starting on the day ofinjection or when tumor volume reached ˜50 mm³.

Cell Culture

Cell lines were obtained from the American Type Culture Collection orthe German Collection of Microorganisms and Cell Cultures. All celllines were tested routinely, and prior to all metabolomic analyses, formycoplasma contamination. RPMI-1640, fetal bovine serum and dialyzedfetal bovine serum (dFBS) were purchased from Invitrogen. Glucose freeDMEM (containing 2 mM Gln), dimethyl αKG, Asp, GSH reduced ethyl ester,OAA, dimethyl malate and DMEM power (without glucose and Gln) wereobtained from Sigma, and Gln-free RPMI 1640 was purchased from Cellgro.Cosmic calf serum (CCS) was obtained from Thermo Scientific. Cells werecultured in the following media: 8988T, Panc1, MPanc96, Miapaca2 andPL45 in DMEM supplemented with 10 mM glucose and 10% CCS; 8902 in RPMIwith 10% CCS; IMR90 in MEM with 10% FBS; HPDE cells were cultured asdescribed previously [Ouyang, H. et al. (2000) Am J Pathol 157,1623-1631]. Primary human PDAC lines were generated from ascites fluidunder IRB approved protocols 02-240 and 2007P001918. The lines wereconfirmed to have Kras mutations by DNA sequencing.

Cell Proliferation Assay

Cells were plated in 24-well plates at 2,000 cells per well in 0.5 mL ofmedia. To deprive Gln, cells were plated in complete culture medium (10mM glucose and 2 mM Gln) which was exchanged with Gln-free culturemedium supplemented with 10% dFBS the following day. Cell culture mediawas not changed throughout the course of the experiment. At theindicated time points, cells were fixed in 10% formalin and stained with0.1% crystal violet. Dye was extracted with 10% acetic acid and therelative proliferation was determined by OD at 595 nm.

Clonogenic Assay

Cells were plated in 6-well plates at 300 cells per well in 2 mL ofmedia. Cell culture media was not changed throughout the course of theexperiment. After 7-10 days, colonies were fixed in 80% methanol andstained with 0.2% crystal violet.

Quantitative RT-PCR

Total RNA was extracted using TRIzol (Invitrogen) and reversetranscription was performed from 2 μg of total RNA using oligo-dT andMMLV HP reverse transcriptase (Epicentre), according to themanufacturer's instructions. Quantitative RT-PCR was performed with SYBRGreen dye using an Mx3000PTM (Stratagene). PCR reactions were performedin triplicate and the relative amount of cDNA was calculated by thecomparative CT method using the 18S ribosomal RNA sequences as acontrol. Primer sequences available upon request.

Oxygen Consumption Rate

Oxygen consumption rates (OCRs) were monitored with the Seahorse XF24instrument (Seahorse Biosciences). 30,000 cells were plated inquadruplicate in a 24-well Seahorse plate in 250 μL of appropriategrowth medium and incubated overnight. Prior to measurements, cells werewashed with unbuffered medium, then immersed in unbuffered medium andincubated in a 37° incubator without CO₂ for 1 hr. Measurements werereported in pmol/min for oxygen consumption.

Xenograft Studies

For subcutaneous xenografts, 8988T cells were infected with lentiviralshRNAs targeting GLUD1 (n=2), GOT1 (n=2), MDH1 (n=2), ME1 (n=2) and GFP(control hairpin, n=1) and subjected to a short puromycin selection (2μg/mL); shRNA ARE sequences below. 1.5×10⁶ cells, suspended in 100 μLHanks Buffered Saline Solution (HBSS), were injected subcutaneously intothe lower flank of NCr nude mice (Taconic). Tumor length and width weremeasured twice weekly and the volume was calculated according to theformula (length×width²)/2. All xenograft experiments with human PDAClines were approved by the HMS Institutional Animal Care and UseCommittee (IACUC) under protocol number 04-605. For mouse xenografts, adoxycycline-inducible GOT1 shRNA construct was first generated. Forgeneration of the construct, oligonucleotides to mouse GOT1 shRNA(forward: CCGGCCACATGAGAAGACGTTTCTTCTCGAGAAGAAACGTCTTCTCATGTGTTTTTG (SEQID NO: 19); reverse: AATTCAAAAACCACATGAGAAGACGTITCTTCTCGAGAAGAAACGTCTTCTCATGTGG (SEQ ID NO: 20)) were digested to generate stickyends (AgeI and EcoRI) and immediately subcloned into the AgeI-EcoRIsites of the pLKO-Tet-on vector. For subcutaneous xenograft, 10⁶ stablyinfected murine PDAC cells were suspended in 100 μl Hanks BufferedSaline Solution and injected subcutaneously into the lower flank of NCrnude mice (Taconic). Animals were fed with doxycycline water(doxycycline 2 g/L, sucrose 20 g/L) starting on the day of injection orwhen tumor diameter reached 50 mm. Tumor volumes were measured everythird day starting from day 4 post-injection and calculated as above.These xenograft experiments were approved under MDACC IACUC protocol111113931.

Western Blot Analysis

After SDS-PAGE, proteins were transferred to Hybond-N Nitrocellulose(Amersham Biosciences). Membranes were blocked in Tris-buffered saline(TBS) containing 5% non-fat dry milk and 0.1% Tween 20 (TBS-T), prior toincubation with the primary antibody overnight at 4°. The membranes werethen washed with TBS-T followed by exposure to the appropriatehorseradish peroxidase-conjugated secondary antibody for 1 h andvisualized on Kodak X-ray film using the enhanced chemiluminescence(ECL) detection system (Thermo Scientific). The following antibodieswere used: Kras (F234, Santa Cruz), GOT1 (NBP1-54778, Novus), GLUD1(ab55061, Abcam) and 3-Actin (A2066, Sigma).

ROS Quantification

The DCFDA assay was performed 24 hr after supplementing Gln-free mediawith either OAA (4 mM) or dimethyl malate (4 mM). Cells were incubatedwith 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Invitrogen)for 30 min. Excess DCFDA was removed by washing the cells twice withPBS, and labeled cells were then trypsinized, rinsed, and resuspended inPBS. Oxidation of DCFDA to the highly fluorescent2′,7′-dichloro-fluorescein (DCF) is proportionate to ROS generation andwas analyzed by flow cytometry.

Metabolomics

For steady state metabolomic analysis, PDAC cell lines were grown to˜50% confluence in growth medium (DMEM, 2 mM Gln, 10 mM glucose, 10%CCS) on 10 cm tissue culture dishes in biological quadruplicate. Acomplete medium change was performed two hours prior to metabolitecollection. To trace Gln metabolism, PDAC cell lines were grown as aboveand then transferred into Gln-free DMEM (with 10 mM glucose) containing10% dialyzed FBS and 2 mM U—¹³C₅-Gln (Cambridge Isotope Labs) overnight(for steady state labeling). Additionally, tissue culture media wasreplaced with fresh media containing [U—¹³C₅]-Gln 2 hr prior tometabolite extraction for steady state analyses. The quantity of themetabolite fraction analyzed was adjusted to the corresponding proteinconcentration calculated upon processing a parallel 10 cm tissue culturedish. Metabolite fractions were collected and analyzed by targetedLC-MS/MS via selected reaction monitoring (SRM), as described [Ying, H.et al. (2012) Cell 149, 656-670; Yuan, M., et al. (2012) Nat Protoc 7,872-881]. Processed data was analyzed using Cluster 3.0 and TreeViewersoftware.

Measurement of Sensitization of PDAC Cells to ROS

PDAC cell lines were plated into 96-well plates at 10³ cells/well in 200μL of growth medium. The following day, growth medium was replaced withthat containing GLS inhibitors and/or H₂O₂. Parallel plates wereanalyzed at 3, 6 and 9 days by Cell Titer Glo analysis (Promega), perthe manufacturer's instruction. The GLS inhibitors 968 (active) and 365(structurally similar, inactive) were provided as a kind gift from theCerione laboratory [Wang, J. B. et al. (2010) Cancer Cell 18, 207-219].BPTES was a kind gift from Jaime Escobedo (Forma Therapeutics,Watertown, Mass.).

Lentiviral-Mediated shRNA Targets

All shRNA vectors were obtained from the RNA Interference ScreeningFacility of Dana Farber Cancer Institute. The RNAi Consortium clone IDsfor the shRNAs used in this study are as follows:

shGLS-1: (SEQ ID NO: 21) GCACAGACATGGTTGGTATAT (TRCN0000051135);shGLS-2: (SEQ ID NO: 22) GCCCTGAAGCAGTTCGAAATA (TRCN0000051136);shMDH1-1: (SEQ ID NO: 23) CCCTGTTGTAATCAAGAATAA (TRCN0000221892);shMDH1-2: (SEQ ID NO: 24) GCAACAGATAAAGAAGACGTT (TRCN0000221893);shME1-1:  (SEQ ID NO: 25) GCCTTCAATGAACGGCCTATT (TRCN0000064728);shME1-2: (SEQ ID NO: 26) CCAACAATATAGTTTGGTGTT (TRCN0000064729);shGLUD1-1: (SEQ ID NO: 27) CCCAAGAACTAFACTGATAAT (TRCN0000220878);shGLUD1-2:  (SEQ ID NO: 28) GCAGAGTTCCAAGACAGGATA (TRCN0000220880);shGOT1-1: (SEQ ID NO: 29) GCGTTGGTACAAFGGAACAAA (TRCN0000034784);shGOT1-2: (SEQ ID NO: 30) GCTAATGACAATAGCCTAAAT (TRCN0000034785);shGPT2-1: (SEQ ID NO: 31) CGGCAFTTCTACGAFCCTGAA (TRCN0000035024);shGPT2-2:  (SEQ ID NO: 32) CCATCAAAFGGCTCCAGACAT; shPSAT1-1:(SEQ ID NO: 33) GCCAAGAAGTTTGGGACTATA (TRCN0000035264); shPSAT1-2:(SEQ ID NO: 34) CCAGACAACTATAAGGTGATT (TRCN0000035265); shKras-1:(SEQ ID NO: 35) CCTCGTTTCTACACAGAGAAA (TRCN0000040148);  and shKras-2:(SEQ ID NO: 36) GAGGGCTTTCTTTGTGTATTT (TRCN0000033260).

Reagents

NADP⁺/NADPH ratios were determined using the NADP/NADPH assay kit(Abcam; ab65349) according to the manufacturer's instructions. Briefly,10⁵ cells (n=6 wells of a 6-well dish) were collected on ice inextraction buffer and subject to two rounds of freeze-thaw at −80° C.NADP+ and NADPH values were determined in biological sextuplet andconcentration was obtained by comparison to standard curves.Mitochondrial fractions were obtained as described previously [Son, J.K., et al. (2010) Cell Death Differ 17, 1288-1301]. OAA was not analyzedby targeted LC/MS-MS due to its limited stability in aqueous solvents atroom temperature [Bajad, S. U. et al. (2006) J Chromalogr A 1125,76-88]. As such, the abundance of this metabolite was determined using aquantification kit (Biovision, Milpitas, Calif.), according themanufacturer's instruction. Briefly, 2×10⁶ cells (n=4 10 cm tissueculture dishes) were collected during log-phase growth bytrypsinization, re-suspended immediately in the buffers provided (onice), analyzed and compared to standard curves. The signals obtainedwere normalized to the protein concentration calculated upon processinga parallel 10 cm tissue culture dish. NEAA mixture consisted of amixture of 0.1 mM glycine, alanine, aspartate, asparagine, proline andserine.

Statistical Analysis

Comparisons were performed using the unpaired Student's t-test. For allexperiments with error bars, standard deviation was calculated toindicate the variation within each experiment and data, and valuesrepresent mean±standard deviation (s.d.).

Example 2 Dependence of PDAC on Glutamine

This example demonstrates that both glucose and Gln are critical forPDAC growth.

To explore the dependence of PDAC on glucose and Gln, and, inparticular, to examine the functional role of Gln in PDAC tumormetabolism, it was first determined whether glucose and Gln wererequired for PDAC growth. As expected from previous work [Ying et al.supra, and U.S. provisional application No. 61/578,116], glucose wasrequired for PDAC growth. Additionally, PDAC cells were also profoundlysensitive to Gln deprivation, indicating that Gln is also critical forPDAC growth (FIG. 1A and FIG. 1B).

Gln supports tumor cell growth through two primary mechanisms: (i) theglutaminase (GLS)-mediated conversion of Gln into glutamate (Glu) allowsfor the use of the Gln carbon skeleton in anaplerotic reactions toreplenish TCA cycle intermediates, and (ii) the side chain amide of Glnprovides nitrogen for nucleotide, nonessential amino acid (NEAA) andhexosamine biosynthesis. To assess the role of Gln metabolism in PDACgrowth, GLS activity was impaired genetically, using RNA interference(RNAi). Notably, GLS knockdown markedly reduced PDAC clonogenic growthand proliferation (FIG. 2 and FIG. 3).

The Gln-derived carbon skeleton of Glu plays an important role inreplenishing intermediates of the TCA cycle that are precursors forbiosynthetic reactions. In particular, Glu can be converted intoα-ketoglutarate (αKG) by glutamate dehydrogenase (GLUD1) or bytransaminases (FIG. 4). Indeed, many cancer cells rely on GLUD1-mediatedGlu deamination to fuel the TCA cycle, and αKG has been shown to be anessential metabolite in Gln metabolism. Therefore, it was next examinedwhether αKG can rescue cell proliferation upon Gln deprivation.Surprisingly, dimethyl αKG (a cell permeable αKG analog) did not restoregrowth upon Gln deprivation (FIG. 5). Thus, the combination of αKG andNEAA, which recapitulates the metabolic output of transaminase-mediatedGlu metabolism, was next tested. This combination dramatically rescuedcell proliferation in multiple PDAC lines (FIG. 5 and FIG. 6),indicating that PDAC cells may metabolize Gln in a manner different fromcanonical models. Furthermore, as transaminases catalyze a reaction thatgenerates both NEAA and αKG, the metabolite rescue data suggested thatthis class of enzymes may be critical for Gln metabolism in PDAC.

Example 3 Identification of a Non-Canonical Pathway of GlutamineMetabolism in PDAC

This example identifies a novel pathway of glutamine metabolism in PDACthat involves the enzymes GOT1, MDH1 and ME1.

PDAC cells were treated with aminooxyacetate (AOA), a potent inhibitorof transaminases [Wise, D. R. et al. (2008) Proc Natl Acad Sci USA 105,18782-18787]. In parallel, Epigallocatechin Gallate (EGCG), anantioxidant and inhibitor of GLUD1, was also utilized. Previously, itwas shown that EGCG (but not AOA) could inhibit the proliferation oftumor cells driven by mTOR activation, indicating that GLUD1 isessential to Gln metabolism in this setting. However in a fibroblastsystem with overexpression of mutant Kras, both transaminases and GLUD1were critical for cell growth. In contrast, it was found that EGCG (FIG.7A) had little effect on PDAC growth, whereas AOA (FIG. 7B) treatmentrobustly inhibited the growth of PDAC cells. Consistent with theseresults, GLUD1 knockdown also had no effect on PDAC growth (FIG. 8).Together, these data indicates that oncogene-induced metabolicalterations are context dependent with PDAC relying predominantly ontransaminases for growth.

Next, to identify the specific transaminases involved in PDAC Glnmetabolism, each of the Glu-dependent transaminases (aspartate, alanineand phosphoserine transaminase) was inhibited individually using RNAiand the effect on PDAC growth was examined. Interestingly, knockdown ofthe aspartate aminotransferase, GOT1, had the most significant impact onPDAC growth (FIG. 8 and FIG. 9), and these observations werereproducible in multiple PDAC cell lines (FIG. 10). As furtherconfirmation of the importance of this pathway in PDAC, GOT1, GLUD1,MDH1, and ME1 expression was suppressed using two lentiviral shRNAs inPDAC cells and the cells' ability to grow as xenografts was assessed.Consistent with in vitro results, both GLUD1 shRNAs had no effect ontumor growth (FIG. 11A). In contrast, GOT1, MDH1, and ME1 knockdown eachrobustly diminished tumor growth (FIG. 11A and FIG. 11B). These dataprovide further support for the critical role of this pathway in Glnmetabolism and PDAC tumor growth.

Next, the direct effects of GOT1 knockdown on Gln metabolism wereexplored. Toward this end, targeted liquid chromatography-tandem massspectrometry (LC-MS/MS) based metabolomic analysis in GOT1 knockdownPDAC cells was performed using uniformly-labeled ¹³C₅-Gln (U¹³-Gln) as atracer [Ying et al. supra; Yuan et al. supra]. GOT1 catalyzes theconversion of aspartate (Asp) and αKG into OAA and Glu in the cytoplasm.Based on the functional data, it was speculated that GOT1 knockdownwould lead to an increase in Asp and a decrease in OAA. Indeed, GOT1knockdown led to increased Gln-derived Asp (and total Asp) and decreasedOAA. Interestingly, a significant decrease in the ratio ofreduced-to-oxidized glutathione (GSH:GSSG) was also observed (FIG. 12and FIG. 13), demonstrating that GOT1 plays a role in the maintenance ofcellular redox homeostasis.

One mechanism by which cells maintain redox homeostasis is through theshunting of glucose into the oxidative arm of the PPP, which generatescellular reducing power in the form of NADPH. In PDAC, it was recentlydemonstrated that glucose is not used to generate NADPH through the PPP,and, moreover, that glucose metabolism has minimal effects on the redoxstate in PDAC [Ying et al. supra, and U.S. provisional application No.61/578,116]. Therefore, it was speculated that the GOT1-mediatedconversion of Gln into OAA, via Asp, may facilitate the downstreamproduction of NADPH.

To test this hypothesis, ROS levels were assessed upon Gln deprivationin the absence or presence of OAA. Indeed, it was found that Glndeprivation induced ROS and that OAA could rescue the elevated ROSlevels caused by Gln deprivation (FIG. 14). Consistent with this result,GOT1 knockdown also increased ROS levels, which again were restored uponmedium supplementation with OAA (FIG. 15). These results demonstratethat GOT1-mediated OAA production from Gln is required for theregulation of ROS.

Given the observation that Gln-derived malate (and total malate) wassignificantly reduced upon GOT1 knockdown (FIG. 12), it was speculatedthat Gln-derived OAA is metabolized into malate, which is ultimatelyutilized by malic enzyme (ME1) to create NADPH, which would providereducing power to maintain pools of reduced glutathione (GSH) necessaryfor redox homeostasis. Consistent with this notion, malate rescued theoxidative stress imposed by Gln-deprivation (FIG. 16) and GOT1 knockdown(FIG. 17). Collectively, these data are consistent with a model wherebyGln-derived Asp is converted by GOT1 into OAA. This OAA is thenconverted into malate by malate dehydrogenase (MDH1) and subsequentlyoxidized by ME1 into pyruvate and NADPH (FIG. 18). Consistent with thispathway, metabolomic analysis of U¹³-Gln tracing in ME1 knockdown cellsrevealed a significant increase in Asp, malate and OAA and decreased GSH(FIG. 19A). Furthermore, knockdown of GOT1 and ME1 markedly increasedthe cellular NADP+/NADPH ratio, whereas inhibition of other cytosolicsources of NADPH (G6PD or isocitrate dehydrogenase, IDH1) had no effecton NADP+/NADPH ratios (FIG. 19B) or ROS. Together the data suggest thatPDAC utilize Gln through the pathway depicted in FIG. 18 to increase theNADPH/NADP+ ratio for maintenance of redox homeostasis. Lastly, Glntracing kinetic flux experiments in GOT1 knockdown cells clearlydemonstrate decreased flux through this pathway. Interestingly, lactatelabeling in the ¹³C-labeling experiments was typically at very lowlevels, indicating that the pyruvate produced by ME1 is not utilized tomake lactate by lactate dehydrogenase.

Example 4 Role of GOT1, MDH1 and ME1 in PDAC

This example demonstrates that GOT1, MDH1 and ME1 activity is requiredin PDAC cells but not normal cells for redox homeostasis and cellproliferation.

The canonical role of GOT1 is to facilitate the shuttling of electronsfrom cytosolic NADH (produced during glycolysis) into the mitochondriafor oxidative phosphorylation. Thus, it was speculated that GOT1knockdown may result in decreased oxidative phosphorylation. To testthis hypothesis, the mitochondrial NADH/NAD⁺ ratio and the oxygenconsumption rate (OCR) were measured. It was observed that both themitochondrial NADH/NAD+ ratio and oxygen consumption were significantlydecreased upon GOT1 knockdown (FIG. 20A, FIG. 20B), demonstrating thatthe GOT1-mediated conversion of Gln-derived Asp into OAA and then malateallows PDAC cells to simultaneously provide NADH for oxidativephosphorylation while generating NADPH to maintain redox homeostasis.The majority of Asp in PDAC cells (50-75%) is derived from Gln, asevidenced by ¹³C-labeling (FIG. 20C). In principle, uniformly¹³C-labeled Asp can be derived from Gln following either (i) theGLUD1-mediated conversion of Glu to αKG (and its subsequent traversingthrough the TCA cycle) or (ii) the mitochondrial aspartate transaminase(GOT2)-mediated conversion of Glu and OAA to αKG and Asp. Of these twoenzymes, only GOT2 knockdown significantly impacted PDAC growth (FIG.20C). Consistent with this observation, GLUD1 knockdown did not affectAsp biosynthesis from Gln, whereas GOT2 knockdown resulted in asignificant decrease in Gln-derived Asp in PDAC cells (FIGS. 20D, 20E).

Since GOT1 is required to support PDAC growth, potentially throughmechanisms involving redox balance, it was next tested whether othercomponents of this pathway are also required to sustain PDAC growth.Indeed, both MDH1 and ME1 knockdown also dramatically inhibitedclonogenic survival of PDAC cells (FIG. 21A, FIG. 21B) in a mannersimilar to GOT1 knockdown (FIG. 8, 11).

The ability of the combination of the GOT1 substrates Asp and αKG torescue PDAC cell growth upon Gln deprivation was assessed. Indeed, thiscombination robustly rescued cell growth in Gln-free conditions (FIG.22). It was speculated that the GOT1-mediated conversion of Gln-derivedAsp into OAA functions in a pathway that is used to generate the NADPHwhich could be used to maintain redox balance. To test this hypothesis,ROS levels were assessed upon Gln deprivation in the absence or presenceof OAA. Gln deprivation induced ROS and OAA could partially rescue theelevated ROS levels (FIG. 23). Additionally, OAA permitted PDAC growthunder Gln-free conditions in multiple PDAC cell lines (FIG. 26) as wellas upon both GLS (FIG. 24) and GOT1 knockdown. GOT1 knockdown alsoincreased ROS levels, which again were significantly restored uponsupplementation with OAA (FIG. 25). Lastly, the addition ofdimethyl-malate was able to partially rescue PDAC cell growth upon Glndeprivation (FIG. 27) or GOT1 knockdown (FIG. 28).

Example 5 PDAC use the GPP to Generate Reducing Power

This example demonstrates that OAA- or malate-mediated rescue of PDACgrowth upon Gln deprivation is via the generation of reducing potential.

Cells grown in Gln-free conditions were treated with cell permeable GSH.Remarkably, the addition of GSH dramatically rescued the clonogenicsurvival of Gln-deprived cells (FIG. 29). Together, these data supportthe idea that Gln is utilized by PDAC cells to produce reducingequivalents, which are required to support continued tumor growth.

In contrast to PDAC, which rely on Gln for redox homeostasis, thispathway appears to be dispensable in normal cells. Indeed, treatment ofnon-transformed human pancreatic ductal cells (HPDE) and human diploidfibroblasts (IMR90) with AOA had no significant effect on growth (FIG.30A, 30B). Interestingly, HPDE cells, unlike PDAC cells, weresignificantly sensitive to EGCG, suggesting a greater reliance on theactivity of GLUD1. Consistent with these results, GOT1 knockdown did notimpair the growth of the normal cell lines HPDE and IMR90 (FIG. 31A,FIG. 31B). Thus, the GOT1-mediated utilization of the Gln carbonskeleton appears to be a metabolic adaptation that PDAC have acquired asa means to generate reducing power. Indeed, this reliance is so completethat knockdown of any component in this pathway disrupts redoxhomeostasis and impairs proliferation in PDAC cells.

Example 6 Role of Oncogenic Kras in PDAC Glutamine Metabolism

This example demonstrates that rewiring of Gln metabolism in PDAC iscontrolled by oncogenic Kras.

It was next investigated whether the rewiring of Gln metabolism in PDACmay be controlled by genetic events that promote PDAC transformation.Mutations in the Kras oncogene are a critical event in PDAC development.Indeed, previous work has demonstrated that glucose metabolism in PDACis controlled by oncogenic Kras, which leads to altered expression of anumber of rate-limiting metabolic enzymes [Ying et al. supra, and U.S.provisional application No. 61/578,116]. To investigate the role of Krasin the metabolic reprogramming of Gln flux, the expression of GOT1 andGLUD1 upon knockdown of Kras in PDAC cells was assessed.

Interestingly, Kras knockdown resulted in a marked increase in GLUD1 anda decrease in GOT1 expression at the transcriptional level (FIG. 32A),as well at the protein level (FIG. 32B) in multiple PDAC lines (FIG.32C). Additionally, using five independent orthotopic tumors derivedfrom the inducible Kras PDAC model described in Ying et al. (supra), itwas determined that expression of GOT1 increased and GLUD1 decreased inan oncogenic Kras-dependent manner in vivo (FIG. 32D). These findingsdemonstrate that, in PDAC, oncogenic Kras plays a critical role incoordinating the shift in Gln metabolism to maintain tumor growth andsurvival.

Additionally, expression of both MDH1 and ME1, enzymes involved in thepathway maintaining the cellular redox state, were significantlydecreased upon Kras knockdown (FIG. 33). These results demonstrate thatin PDAC, oncogenic Kras plays a critical role in coordinating this shiftin Gln metabolism to maintain tumor growth and survival. While it hasbeen shown previously in other tumor types that transformation withoncogenic Ras promotes Gln uptake to balance redox demands, this occursthrough mechanisms that are distinct from the pathway shown herein to beutilized in PDAC [Weinberg, F. et al. (2010) Proc Natl Acad Sci US A107, 8788-8793; Trachootham, D. et al. (2006) Cancer Cell 10, 241-252;DeBerardinis, R. J. et al. (2007) Proc Natl Acad Sci USA 104,19345-19350]. In addition, there may also be context-dependent effectson Gln metabolism, as different oncogenic drivers also have roles inpromoting Gln metabolism in other tumor types [Ward, P. S. & Thompson,C. B. (2012) Cancer Cell 21, 297-308; Wise et al. supra; Gao, P. et al.(2009) Nature 458, 762-765].

Next, the sensitivity of PDAC cells to either AOA or EGCG upon Krasknockdown using 8988T cells, a cell line that is not dependent on Krasfor survival [Singh, A. el al. (2009) Cancer Cell 15, 489-500], wasassessed. Consistent with previous results, AOA significantly inhibitedclonogenic growth, whereas EGCG had minimal effects. Interestingly, Krasknockdown made the cells significantly more resistant to AOA andsensitive to EGCG (FIG. 34).

To confirm the role of oncogenic Kras in the reprogramming of Glnmetabolism, a targeted metabolomic analysis using U¹³-Gln was performedto characterize alterations upon Kras knockdown. Indeed, the changesobserved were consistent with Kras-supporting the anabolic metabolism ofGln, where multiple metabolites in the GOT1-dependent pathway mediatingNAPDH production were significantly deregulated in a manner consistentwith the proposed pathway (FIG. 35).

Given the importance of Gln metabolism in maintaining the redox state ofPDAC, it was speculated that low Gln and/or Glutaminase (GLS) inhibitionmay sensitize PDAC to oxidative stress. To test this concept, Glnmetabolism was inhibited in PDAC cells using a GLS inhibitor and it wasexamined whether this would synergize with hydrogen peroxide (H₂O₂)treatment. Indeed, two chemically distinct GLS inhibitors (968/365(active/inactive form) or BPTES) [Wang et al., supra; DeLaBarre, B. etal. (2011) Biochemistry 50, 10764-10770] had a growth suppressive effecton PDAC cells, consistent with the GLS knockdown data (FIG. 36).Furthermore, when combined with H₂O₂, this effect was dramaticallyaugmented, indicating that PDAC cells are markedly more sensitive to ROSwhen Gln metabolism is impaired (FIG. 37 and FIG. 38). This finding hassignificant therapeutic implications, as clinical grade GLS inhibitorsare being developed [Vander Heiden, M. G. (2011) Nat Rev Drug Discov 10,671-684], and standard PDAC therapies (such as radiation) lead to thegeneration of ROS. Moreover, since this aspect of Gln metabolism doesnot appear as critical in normal cells, these data suggest attractivetreatment combinations with an accessible therapeutic window forpatients with PDAC.

Collectively, the present Examples demonstrate that PDAC utilizes Gln togenerate OAA via GOT1, and, sequentially, this OAA is converted intomalate and then pyruvate. This series of reactions results in thetransport of NADH into the mitochondria (for oxidative phosphorylation),while simultaneously providing the reducing power necessary to sustainPDAC growth and survival through reducing equivalents generated by ME1upon conversion of malate into pyruvate. Importantly, oncogenic Krasappears to support this pathway through the regulation of expression ofkey metabolic enzymes, and this reprogramming of Gln metabolism isindispensable for tumor maintenance in PDAC (FIG. 39).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. It isfurther to be understood that all values are approximate, and areprovided for description. Accordingly, other embodiments are within thescope of the following claims.

1. A method for determining the efficacy of glutamine to pyruvatepathway (GPP)-targeting in a subject comprising an oncogenic Krasmutation, and the GPP-targeting comprises targeting an enzyme ormetabolite associated with an enzyme-catalyzed reaction in the GPP thatis upstream of the malic enzyme (ME1)-catalyzed reaction, the methodcomprising: determining the level of one or more markers selected fromthe group consisting of NADP+, NADPH, GSSG, GSH, pyruvate, and reactiveoxygen species (ROS), in a sample obtained from a subject who isundergoing or has undergone the GPP-targeting; and concluding that theGPP-targeting was effective if the level of one or more of the markersNADP+, GSSG and ROS is increased, relative to each marker's controllevel, or if the level of one or more of the markers NADPH, GSH andpyruvate is decreased, relative to each marker's control level; orconcluding that the GPP-targeting was not effective if the level of oneor more of the markers NADP+, GSSG, and ROS is not increased, relativeto each marker's control level, or if the level of one or more of themarkers NADPH, GSH and pyruvate is not decreased, relative to eachmarker's control level. 2-6. (canceled)
 7. The method of claim 1,wherein each marker's control level is the level of the marker in asample obtained from the same subject prior to or at the beginning ofthe GPP-targeting or from another subject who is known to comprise anoncogenic Kras mutation and is not undergoing or has not undergoneGPP-targeting.
 8. The method of claim 1, wherein each marker's controllevel is a predetermined reference level of the marker.
 9. (canceled)10. The method of claim 1, wherein the GPP-targeting inhibits one ormore of the enzymes selected from the group consisting of Kras, GLS,GOT1, GOT2 and MDH1.
 11. The method of claim 10, wherein the methodfurther comprises determining the level of at least one additionalmarker selected from the group consisting of glutamine, glutamate,aspartate, ctKG, NAD+, NADH, oxaloacetate, malate, MDH1, and ME1. 12.The method of claim 1, wherein the targeting of an enzyme comprisesinhibition by an inhibitor selected from the group consisting of ananti-sense oligonucleotides, shRNA, siRNA, intrabodies, and a smallmolecule. 13-16. (canceled)
 17. A method for determining the efficacyofoncogenic Kras inhibition in a subject comprising a cell having anoncogenic Kras mutation, the method comprising: determining theexpression level of at least one of the enzymes MDH1 and ME1 in a sampleobtained from a subject who is undergoing or has undergone the oncogenicKras inhibition; and concluding that the oncogenic Kras inhibition waseffective if the expression level of the at least one enzyme isdecreased compared to a control level of the enzyme; or concluding thatthe oncogenic Kras inhibition was not effective if the expression levelof the at least one enzyme is not decreased compared to a control levelof the enzyme.
 18. The method of claim 1, wherein the subject has beendiagnosed with a cancer associated with an oncogenic Kras mutation.19-24. (canceled)
 25. The method of claim 1, wherein the oncogenic Krasmutation is selected from the group consisting of Kras^(G12D),Kras^(G12V), Kras^(G13D), Kras^(G12C), Kras^(Q61R), Kras^(Q61L),Kras^(Q61K), Kras^(G12R), and Kras^(G12C).
 26. (canceled)
 27. The methodof claim 18, wherein the cancer associated with an oncogenic Krasmutation is selected from the group consisting of pancreatic cancer,non-small cell lung cancer, colorectal cancer, and biliary cancer. 28.(canceled)
 29. A method for determining the efficacy of glutamine topyruvate pathway (GPP)-targeting in a subject comprising an oncogenicKras mutation, and the GPP-targeting comprises targeting one or more ofthe enzymes catalyzing the conversion of glutamine to aspartate, themethod comprising: determining the level of at least one marker selectedfrom the group consisting of GSSG, GSH, aspartate, αKG, NADP+, NADPH,NAD+, mitochondrial NADH, pyruvate, oxaloacetate, and malate in a sampleobtained from a subject who is undergoing or has undergone theGPP-targeting; and concluding that the GPP-targeting was effective ifthe level of one or more of GSSG, GSH, aspartate, αKG, NADP+, NADPH,NAD+, NADH, pyruvate, oxaloacetate, and malate is altered relative toeach marker's control level.
 30. (canceled)
 31. A method for treatingcancer in a subject comprising a cancer cell comprising an oncogenicKras mutation, the method comprising administering to the subject: (i) atherapeutically effective amount of a composition comprising aninhibitor of the glutamine to pyruvate pathway (GPP) enzyme ME1; or (ii)a therapeutically effective amount of an inhibitor of the enzyme GLS orGOT2; and a therapeutically effective amount of an inhibitor of one ormore the enzymes selected from the group consisting of Kras, GOT1, MDH1,and ME1, or a therapeutically effective amount of an inhibitor of atleast one metabolite or other compound associated with anenzyme-catalyzed reaction in the glutamine to pyruvate pathway (GPP).32-34. (canceled)
 35. The method of claim 31, wherein the oncogenic Krasmutation is selected from the group consisting of Kras^(G12D),Kras^(G12V), Kras^(G13D), Kras^(G12C), Kras^(Q61R), Kras^(Q61L),Kras^(Q61K), Kras^(G12R), and Kras^(G12C).
 36. (canceled)
 37. A kitcomprising: an inhibitor of ME1 and one or more inhibitors of one ormore of the enzymes selected from the group consisting of Kras, GOT2,GLS, GOT1, and MDH1; an inhibitor of GLS and one or more inhibitors ofone or more of the enzymes selected from the group consisting of Kras,GOT2, ME1, GOT1, and MDH1; an inhibitor of at least one of the enzymesselected from the group consisting of Kras, ME1, GLS, GOT1, GOT2, andMDH1, and an inhibitor of one or more metabolites associated with anenzyme-catalyzed reaction in the glutamine to pyruvate pathway (GPP); oran inhibitor of two or more metabolites associated with anenzyme-catalyzed reaction in the glutamine to pyruvate pathway (GPP).38-42. (canceled)
 43. A method for preventing cancer in a subjectcomprising an oncogenic Kras mutation, the method comprisingadministering to the subject: (i) a therapeutically effective amount ofa composition comprising an inhibitor of the glutamine to pyruvatepathway (GPP) enzyme ME1; or (ii) a therapeutically effective amount ofan inhibitor of the enzyme GLS or GOT2, and a therapeutically effectiveamount of an inhibitor of one or more the enzymes selected from thegroup consisting of Kras, GOT1, MDH1, and ME1, or a therapeuticallyeffective amount of an inhibitor of at least one metabolite or othercompound associated with an enzyme-catalyzed reaction in the glutamineto pyruvate pathway (GPP). 44-46. (canceled)